James Piret

Professor

Relevant Thesis-Based Degree Programs

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Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Bioprocess and cell line engineering to improve pancreatic endocrine differentiation outcomes and increase safety of pluripotent stem cells (2023)

Diabetes is a debilitating disorder that affects nearly 500 million people worldwide and is characterized by chronic hyperglycemia caused by pancreatic β-cell dysfunction or death. Those that are affected need to frequently monitor their blood glucose levels and administer exogenous insulin, but this strategy does not match the tight regulation maintained by healthy β-cells. Progress in the field has demonstrated an effective clinical path to treat type 1 diabetes through the success of islet transplants. More recently, groups have demonstrated that human pluripotent stem cells, differentiated into pancreatic progenitors or beyond, can prevent or reverse diabetes in rodents and have shown promising results in human clinical trials. Here I develop a differentiation protocol to further differentiate pancreatic progenitors generated with a commercially available kit into endocrine cells. I identified FGF7 signalling as being important for aggregate survival and Notch-inhibition improved endocrine commitment at the expense of ductal cells. Combining in vitro β-cell differentiation strategies with newly developed precision gene editing techniques allows for disease modelling in a dish and transgene elements can be introduced into cells to provide new functionalities. To this end, I used a novel gene editing technique to correct a single-base mutation in pluripotent stem cells derived from an individual living with a severe form of neonatal diabetes. Using stem cell-derived cell therapies does not come without risk, given the potential for teratoma outgrowth, inappropriate differentiation and function, or an immune response to the graft. Here, I generated cell lines containing an inducible safety-switch, which was shown to eliminate pluripotent and pancreatic progenitor cells in vitro and teratomas in vivo. Finally, I targeted the B2M locus for silencing using an inducible CRISPRi platform, preventing HLA-ABC formation in a reversible manner. This strategy could both allow cells to avoid immune detection as well as act as a safety-switch by deactivating CRISPRi and re-expressing the B2M gene. Overall, the projects described in this dissertation demonstrate the combined potential of pluripotent stem cells, gene editing, and cell differentiation with the goal of advancing the cell therapy field, particularly for diabetes.

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Engineering Chinese Hamster Ovary cell recombinant protein glycosylation (2023)

Since 2015 more than 39 biosimilars have been approved by the FDA, with over 20 launched, providing alternatives for widely used protein therapeutics. A major challenge in biosimilar development is the genetic differences in the host cell lines used to manufacture these biologics. Many biologics approved before 2011 were expressed in murine cell lines, whereas Chinese Hamster Ovary (CHO) cells have become the preferred hosts for production. Differences between glycosylation have been identified in biologics produced using murine CHO cell line. In the case of monoclonal antibodies (mAbs), differences in glycans can significantly impact critical antibody effector function, binding activity, efficacy, and in vivo half-life. To more closely match the reference biologic (RB) glycosylation, a mAb expressing CHO cell line was genetically engineered to express mAb with murine-like glycans. This was done by selectively co-expressing two key murine enzymes, α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-Acetylneuraminic acid hydroxylase (CMAH). These CHO cells were then analyzed using a spectrum of analytical methods to demonstrate biosimilarity, including high-resolution mass spectrometry and biochemical and cell-based assays. In fed-batch cultures, two CHO cell clones were identified that maintained stable production while matching the glycosylation profile and function of the reference product. This study demonstrates the feasibility of engineering CHO cells to express mAbs with murine glycans, to reduce the residual uncertainty regarding biosimilarity, resulting in a higher probability of regulatory approval and potentially reduced costs and time in development. In parallel, a high-throughput CHO-specific assay was designed and evaluated to quantify N-glycosylation-related enzyme mRNA expression levels. This assay is referred to as the N-glycan enzyme mRNA expression profiler. Two different mAbs produced by two CHO cell hosts were investigated to assess the relationships between glycan mRNA levels and mAb glycan profiles. Genetic understanding of the N-glycosylation pathway in the CHO cells helped identify gene expression patterns that correlated to the N-glycan profile of the produced mAb. The results indicated that this assay could identify clones with desirable genetic makeups early in cell line development and aid in designing cell line engineering strategies to obtain the desired glycan profiles for both novel biologic and biosimilar development.

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Glutamine and glutamine substitute influences on Chinese Hamster Ovary cell fed-batch culture processing (2023)

The majority of monoclonal antibodies (mAbs) are produced by fed-batch processes using Chinese Hamster Ovary (CHO) cells. Although glutamine has been typically provided in excess, its metabolism and decay cause ammonium accumulation, a toxic byproduct that can inhibit cell growth and reduce culture productivity. To understand how best to reduce ammonium accumulation, both the partial and total replacement of glutamine by asparagine were investigated. The CHO-K1 cells grew at the highest rate in batch cultures when both asparagine and glutamine were supplied, indicating that asparagine can contribute to maximal cell growth rates. Without glutamine in the medium, we confirmed that cultures with asparagine were able to grow at intermediate rates and without increasing cell death. However, without both asparagine and glutamine, or with asparagine and an inhibitor of the glutamine synthesis pathway, the CHO-K1 cells did not grow, and cell death was increased. As a result, it was concluded that the growth of CHO-K1 cells in this study required both glutamine synthetase activity and asparagine when glutamine was not included in the medium. These findings were then tested in industrially relevant fed-batch cultures. When glutamine was either not fed or also not supplied in the initial medium, culture growth was unaffected, and the culture viability was increased. Furthermore, the specific productivity of monoclonal antibody was 1.5-fold higher. These improvements in culture physiology were also studied by metabolomics and transcriptomic analyses. The changes in metabolome levels were more notable than changes in transcript levels. The improvements in the cultures without glutamine in the medium could be attributed to substrate-level control of metabolic reactions rather than significant changes in transcriptomic gene expression. This study advanced our understanding of the physiology of CHO-K1 cells in batch and fed-batch conditions and substantiated a promising prospect for “glutamine-free” fed-batch cultures that increased the mAb productivity of CHO cells. It also revealed challenges in setting up small scale experimental cultures at pre-specified pH levels in bicarbonate-buffered cell culture medium formulations, such that a model was developed and tested for the a priori formulation of these widely used media.

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Bioprocess engineering of cell encapsulation (2022)

Encapsulated beta cell replacement therapies can restore blood glucose control and reduce the need for immunosuppression, and thus should become an attractive alternative for millions of people who currently treat their diabetes with insulin. This work describes two approaches to immobilize pancreatic cells in alginate using: (i) microencapsulation by emulsification and internal gelation in stirred vessels, and (ii) macroencapsulation in fibres by co-axial flow-focusing bioprinting. Scalable generation of microspheres sized 300-800 µm from 5% alginate solutions and higher cell encapsulation yields were attained using grid impellers and by reducing alginate viscosity through thermal treatments. The encapsulation yield was 2-fold higher for 11-µm cells compared to 110-µm aggregates. Also, large numbers of empty microspheres were observed after aggregate encapsulation, likely due to their stochastic distribution in the alginate beads. Microspheres containing aggregates were separated by sedimentation in a continuous density gradient that removed ~50% of the empty bead volume, followed by centrifugation to remove unencapsulated cells. Separation based on density differences could be adapted for other cell microencapsulation technologies by tuning the continuous density gradient as a function of volume fraction per bead. The last part of this thesis, using an analytical mass transfer model, describes the design of cell-laden multi-layered fibres based on avoiding oxygen limitations. Radial displacement of the cell-containing layer, obtained by constraining it between two acellular alginate layers to form an annulus, can increase cell loading per unit length of fibre without compromising cell oxygenation and insulin secretory capacity. This novel bioprinted fibre geometry could be used to encapsulate insulin-producing cells in retrievable devices and leverage oxygenation via passive diffusion as an option to develop carefree alternatives for reversal of diabetes.

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Bioprocessing optimization to manufacture thymus-derived regulatory T cells for therapy (2022)

Regulatory T cell therapy has shown promise in treating autoimmune disorders, transplant rejection and graft-versus-host disease in early clinical trials. However, efficient manufacturing of clinical grade cells is still a significant hurdle that must be overcome before these therapies can see widespread use. Previous work showed that large numbers of pure, naïve Tregs can be isolated from pediatric thymus. This research aims to investigate the variables governing Treg expansion with serum-free media and non-cell-based activation reagents to develop manufacturing protocols to produce therapeutic doses of thymus-derived Tregs. First, we tested activation reagents, cell culture media, restimulation timing, and cryopreservation to develop good manufacturing practice compatible protocols to expand and cryopreserve Tregs. Cryopreservation tests revealed a critical effect of timing: only cells cryopreserved 1-3 days, but not > 3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. We next investigated how changing cell density and feed frequency influenced Treg expansion, viability, and phenotype in a 3-week expansion protocol and found that Treg viability and expansion were correlated with the cell density at restimulation. Tregs restimulated at low cell densities (1x10⁵ cells/cm²) initially had high growth rates, viability, and FOXP3 expression, but at later culture times these parameters were reduced compared to slower growing Tregs restimulated at higher cell densities (5x10⁵ cells/cm²). High density expansion was associated with lower nutrient concentrations and higher accumulations of lactate, but this could be alleviated by decreasing the interval between feeds. We tested platforms to scale up Treg manufacturing and observed that Tregs expanded in gas permeable cell expansion bags were of higher quality than those expanded in agitated suspension culture or the G-Rex. Finally, we tested labelling expanded Tregs with a ¹⁹F-perfluorocarbon (PFC) nanoemulsion to enable in vivo tracking using MRI. While Tregs could be labelled with the ¹⁹F-PFC and detected in vivo in immunocompromised mice, labelling during expansion reduced cell viability, particularly after cryopreservation. Together, this research developed protocols and process understanding necessary to efficiently produce clinical grade Tregs, laying the groundwork for the first clinical trial of thymic Treg cell therapy in Canada.

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Improving the efficacy of hemostatic agents by increasing their transport into wounds (2018)

Uncontrollable hemorrhage remains a leading cause of mortality in many situations, including during surgery and following trauma. Many hemostatic interventions have been developed, but mortality and morbidity remain high because they are ineffective or difficult to use in cases of severe bleeding. Blood flow can rapidly wash away topically applied hemostatic agents and prevents them from reaching leaking vessels and forming robust clots. We hypothesized that increasing the transport of hemostatic agents into wounds could improve their ability to manage bleeding. To test this hypothesis, I developed self-propelling particles that can transport through flowing blood and into wounds. These particles, consisting of calcium carbonate and an organic acid, delivered two hemostatic agents, thrombin and tranexamic acid (TXA), and improved their ability to stop bleeding in mice (Chapter 3). Next, I showed that self-propelling particles loaded with thrombin applied with gauze (PTG) significantly improved survival in a swine model of massive traumatic bleeding without compression, compared to those agents on gauze without propulsion (Chapter 4). This demonstrates that increasing transport of hemostatic agents could increase their utility for managing clinically relevant hemorrhage, such as from battlefield trauma. In two sheep models of surgical hemorrhage, PTG’s ability to stop bleeding was compared to two clinical standard interventions (Chapter 5). In a model of endoscopic surgical bleeding, PTG significantly reduced bleeding time compared to control gauze. In a model of massive open surgical bleeding, PTG achieved hemostasis in more cases than a standard thrombin-containing hemostatic agent. These demonstrate that increased transport of hemostatic agents could enable improved management of surgical bleeding. Finally, formulating TXA with self-propelling particles increased its ability to inhibit fibrinolysis in vitro and reduce bleeding in mice in vivo, demonstrating that these particles could transport a variety of hemostatic agents and increase their efficacy too (Chapter 6). The findings of this thesis suggest that improving the transport of hemostatic agents into wounds, such as by using self-propelling particles, is a promising approach for increasing the efficacy of those agents, and may present an opportunity to reduce mortality, morbidity, and the clinical burden associated with bleeding.

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Small volume cell culture technology for the analysis of clonal heterogeneity in mammalian cell populations (2017)

The ability to culture individual cells provides a unique method to assess the heterogeneity of mammalian cell populations. However, there are many challenges when scaling down culture systems due to the complexity of re-creating a stimulating environment at the clonal level. Small volume culture systems such as integrated microfluidic platforms offer the potential to radically alter the throughput of clonal screening through the use of time-lapse imaging, dynamic stimulus control and economy of scale. In particular, the use of automated fluidic control allows for the characterization of single cells in a dynamic microenvironment similar to large-scale culture. This thesis describes how small volume cell culture practices such as the use of conditioned medium and microfluidic technology can be implemented to isolate large numbers of cells in small volumes and evaluate clonal populations under precise medium conditions. For a Chinese Hamster Ovary (CHO) cell system normal growth kinetics and specific productivity were sustained in small volumes. When exposed to conditioned medium from a parental CHO line, clones cultured at sub-mL scales matched the performance of large-scale cultures. A microfluidic bead assay was developed to detect Immunoglobulin G titers secreted from clones in nL volumes. The combination of microfluidic conditioned medium perfusion with the magnetic bead assay allowed for clonal productivity to be evaluated under simulated fed-batch conditions. Lastly, microfluidic cell culture was demonstrated on a human embryonic stem cell (hESC) system through the robust generation of colonies derived from single cells. hESCs propagated in the microfluidic system were observed to match the growth kinetics, marker expression and colony morphologies of larger cultures, while resolving response heterogeneity during differentiation induction. This thesis demonstrates how high-throughput, small volume culture systems can be used to screen clonal populations for therapeutic applications under complex culture conditions.

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Development of a novel histone-based reagent to increase retroviral transcution based on an analysis of cell lysate effects (2013)

Recombinant retroviruses are widely used vectors for engineering cells as they facilitatethe delivery and long-term integration of transgenes. Retrovirus-mediated gene transfer isnonetheless challenged by low efficiency due to multiple extracellular rate-limiting stepsincluding mass transport limitations due to the low diffusivity and rapid decay ofretroviral vectors as well as limited binding due to electrostatic repulsion between the netnegative-charge of both target cells and retroviral vectors. Whole cell lysate wasdetermined to contribute to the variability observed in transduction protocols leading toan increase in the retroviral transduction of TF-1 (human) cells by gibbon ape leukemiavirus-pseudotyped vectors and of BaF3 (mouse) cells by ecotropic retroviral vectors.Fractionation of the cell lysate revealed that the bulk of the activity was associated withdebris aggregates. These aggregate structures enhanced transduction by increasing masstransport through sedimentation as well as by increasing adsorption of the retroviralvectors to the cells and the culture vessel surfaces. In the presence of this aggregatefraction, transductions of TF-1 and BaF3 cells were enhanced respectively by 59- and213-fold relative to controls without additives. Further analysis revealed that theaggregate structures were derived from nuclear components sensitive to trypsin digestion,suggesting that nuclear proteins rich in arginines and/or lysines were responsible for theobserved enhancement. A subsequent investigation of histone proteins revealed that thearginine-rich fraction, at a concentration of 160 μg/mL, yielded a 22-fold increase in thetransduction of TF-1 cells. To mimic the aggregate structures observed in the lysate,histone self-aggregation was stimulated by heat treatment resulting in a 34-fold increasein TF-1 cell transduction while the concentration required was reduced to 10 μg/mL.With BaF3 cells, the transduction exceeded that achieved with lysate under similarconditions. This reagent was also successfully applied to the transduction of primarymouse hematopoietic progenitor cells with long-term reconstitution potential. Overall, anovel histone reagent able to enhance the transduction of primary cells and cell lines withnegligible toxicity using both gammaretroviral and lentiviral vectors was designed byisolating the active compounds and analyzing the mechanism of action of lysates onretroviral transduction.

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Microfluidic cell culture arrays for clonal expansion and characterization of mammalian cells (2013)

Single-cell culture provides a unique means to reveal the heterogeneitywithin mammalian cell populations. Advances in multilayer soft lithographyhave enabled the development of high-throughput nanoliter-volume cellculture platforms with integrated and programmable fluidic control toprecisely modulate the microenvironment. Coupled with time-lapse imaging,these microfluidic systems allow hundreds of single cells to be monitoredsimultaneously while providing analytical advantages to characterize eachclone. However, there are many challenges associated with theminiaturization of mammalian cell cultures and even greater difficulties fornon-adherent cell types. This work shows how microfluidic devices and theircontrol system can be designed to gently trap suspension cells and enablerobust clonal expansion. Mouse hematopoietic stem cell (HSC) populationswere chosen for their sensitivity and stringent cell culture requirements todemonstrate that normal cell growth and function could be sustained in themicrofluidic system. Using microfluidic clonal analysis and image processingit was observed that cells from HSC-enriched populations had highlyheterogeneous growth profiles. Automated medium exchange and temporalstimulation were then exploited to show that a high Steel factor (SF)concentration was needed for survival of primary HSCs specifically at thetime of exit from quiescence. The ability to perform live immunostaining wascombined with genealogical tracing to identify distinct characteristics, suchas long cell cycle times and frequent asynchrony of daughter cells, associatedwith HSC clones exhibiting persistent endothelial protein C receptorexpression (EPCR) after in vitro culture. Finally, the flexibility of thismicrofluidic system was demonstrated with the culture of Chinese hamsterovary (CHO) cells, the most widely used suspension-adapted mammalian celltype for the production of therapeutic recombinant proteins. In this system, the high cell density and the rapid concentration of cell-secreted products innanoliter-volume chambers were exploited to measure the amount of secretedmonoclonal antibodies from single cells and to increase their cloningefficiency. The ability to recover clones from the microfluidic system hasallowed the selection and expansion of high-producing cell lines. This thesisdemonstrates the potential and adaptability of high-throughput microfluidicsingle-cell culture systems for both research and therapeutic applications.

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Modulating Autophagy and Glutamine Metabolism in CHO Cells to Increase Fed-batch Process Performance (2012)

Valuable recombinant therapeutic proteins are routinely produced from Chinese hamster ovary (CHO) cells in fed-batch cultivations. An improved understanding of the physiological factors that affect cell proliferation, survival, productivity and product quality in fed-batch could contribute to facilitate general access to these products. This work describes the investigation of autophagy and glutamine metabolism in CHO cells for the purpose of increasing fed-batch process performance. The close link between glutamine deprivation and autophagy was found to greatly affect process performance, with an increase of the cellular lysosomal compartment correlated with decreased cell-specific productivity. The increased autophagic activity upon glutamine withdrawal was confirmed by the formation of GFP-LC3 fluorescent puncta and by an LC3 autophagic flux assay. The use of 3-methyl adenine (3-MA) to inhibit autophagy increased the yield of recombinant tissue plasminogen activator (t-PA) by 2.8-fold, without compromising the glycosylation capacity of the cells given that the t-PA fucosylation, galactosylation and sialylation all increased. A more comprehensive study of glutamine metabolism and autophagy performed, including by investigating 2 additional CHO cell lines expressing different antibody proteins. The mitochondrial and lysosomal changes in response to glutamine deprivation varied substantially between cell lines, illustrating how the susceptibility to autophagy can be cell-line dependent. Integrating the combined effect of enhanced proliferation (achieved through modulation of glutamine metabolism) and inhibition of autophagy (by treatment with 3-MA), a maximum 4.6-fold increase of t-PA production was obtained in fed-batch culture. Finally, autophagy and glutamine metabolism were explored in cancer cell lines, and produced original findings on the potential for Raman spectroscopy to analyze live cell physiological responses to conditions that trigger autophagy. Overall, this study illustrates the potential for a fruitful interaction between basic scientific research and applied biotechnology. The investigation of response mechanisms to cellular stress provided opportunities to both improve industrial processing and open new perspectives for basic biological research.

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Bioprocess development for the cell-based treatment of diabetes (2011)

The widespread cellular treatment of type 1 diabetes by islet transplantation islimited by tissue shortage and graft rejection. This work describes two novel bioprocesses toimmobilize pancreatic cells in alginate using: (1) hollow fiber bioreactors or (2) alginate beadgeneration by an adapted emulsion and internal gelation process. After optimization, livecell recovery rates and growth rates were not significantly different between these morescalable processes and conventional methods of alginate immobilization. After 10 days ofalginate-immobilized culture, the insulin content of neonatal porcine cells cultured in ahollow fiber bioreactor increased by 4 fold, while the insulin expression of human isletdepletedtissue cultured in emulsion beads increased by 67 ± 32 fold, matching previousreports that used small-scale cultures. Solutions with >100 Pa·s viscosity could be used withthe emulsion process to generate beads with higher concentration and greater antibodyexclusion than has been so far permitted by nozzle-based encapsulators. The 5% alginatebeads generated by the emulsion process led to blood glucose normalization of allogeneic β-cells transplanted into diabetic mice within 2 weeks, while mice transplanted with 1.5%alginate beads generated by a conventional encapsulator remained hyperglycemic after 20days. The improved result with the 5% alginate emulsion beads was associated with lowergraft-specific antibody plasma levels. These results suggest that the 5% alginate beadsprovided improved immune isolation of the graft. If human pancreatic progenitors are to beused for the large-scale generation of insulin+ cells in alginate, their expansion in serum-freemedium will be a prerequisite. Pancreatic duct-like cells are expected to have more potentialto generate insulin+ cells than the fibroblast-like cells that overgrow unsorted cultures ofislet-depleted human pancreatic tissue. The last part of this thesis describes the magneticactivateddepletion of CD90-expressing cells, which reduced the fraction of CD90+fibroblast-like cells from 34 ± 20% to 1.3 ± 0.6%. This allowed the expansion of the duct-likecell population in an optimized serum-free medium. These novel pancreatic cell culturemethods could be used to generate and/or offer immune protection to insulin+ cells for theclinical-scale cellular treatment of diabetes.

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Characterization and modulation of adult pancreatic beta-cell maturity (2010)

No abstract available.

Increasing the performance of mammalian perfusion culture system (2010)

Perfusion culture processes are potentially the most efficient way of producing large quantities of biopharmaceuticals. However, these processes are not the most commonly used by industry in part due to a lack of simple solutions to perfusion challenges. This thesis has investigated recombinant protein production instability, a strategy to improve low perfusion rate culture performances and the complications due to cell aggregate formation. Human embryonic kidney 293 (HEK293) cells producing recombinant human interferon-alpha2b (IFN-α2b) were investigated as a model recombinant cell line. These cells also were maintained for more than 4 weeks in batch cultures using three media with different osmolalities in order to evaluate production stability. Exposure to high osmolality (~ 375 mOsm kg­‾¹) gradually decreased the yield of IFN-α2b secreted by the viable cells. Perfusion cultures validated the batch cultures with results showing that it was not possible to maintain stable production at elevated osmolality whereas at normal osmolality (~ 300 mOsm kg­‾¹), the titer was maintained at 250 mg L­‾¹. A reduced perfusion rate strategy was explored to increase the product titer in a perfusion bioreactor using enriched media. The HEK293 cell line was found to have a growth-associated production. By increasing the bleed rate in order to increase growth, the perfusion process yielded an up to 35% increased IFN-α2b concentration. Several modified medium conditions were investigated in batch cultures to help identify the main mechanism of aggregation observed in perfusion cultures. The addition of dead cells in the batch case was found to yield cellular aggregation that was most similar to perfusion cultures. The presence of aggregation did not affect the on-line monitoring of the viable cell concentration using a permittivity signal. However, if cells in aggregates are neglected, this can result in major cell specific-rate calculation errors. The image analysis used to estimate the cellular content of aggregates was an efficient method of improving viable cell estimates and cell specific-rate analyses. Overall, advances in the methods to more efficiently monitor and operate high performance perfusion-culture processes should expand their potential to fulfill the increasing demand for recombinant protein products from biotechnology.

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Quantitative analysis of retrovirus-mediated gene transfer into mammalian cells (2010)

Recombinant retroviruses (gammaretro-, lenti- and foamy-viral vectors) are used for gene therapy as well as for scientific research because of their ability to provide relatively stable gene transfer and expression. The process of retrovirus-mediated gene transfer into mammalian cells involves a series of transduction events that take place sequentially. A mathematical model for the retroviral transduction process was developed that incorporates the important extracellular and intracellular rate-limiting steps. The mathematical model was validated with experimental data obtained using gibbon ape leukemia virus envelope pseudotyped retroviral vectors and K562 target cells. The model predictions of transduction efficiency and integrated virus copy number were generally in good agreement with measured results acquired for both static and centrifugation-based gene transfer protocols. However, a deviation between the model calculations and the experimental transduction efficiency data was observed for centrifugation at high vector-to-cell ratios. To address this limitation, believed to be caused by the saturation of binding sites on the cell surface, a detailed experimental investigation of the binding and entry kinetics of retroviruses was performed. With the help of a mathematical representation for retroviral binding to, dissociation from and entry into mammalian cells, the kinetic rate constants for these three steps were experimentally quantified. The model was modified to incorporate these more complex kinetic steps and was shown to provide even better predictions of the experimental transduction results for the full range of vector-to-cell ratios investigated. The modified model was then used to optimize various retroviral transduction process parameters. Studies of the extracellular transport of viral vectors provided the optimal range of centrifugal forces needed to obtain the maximum transduction efficiency. Based on a simulated performance comparison of the three different types of recombinant retroviruses, lentiviral vectors were found to be very efficient for targeting hematopoietic stem cells. The mathematical model was able to provide a set of conditions where retroviral transduction protocols should yield high transduction efficiencies while maintaining the viral copy number in a lower, more desirable range.

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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Novel activity assay development for soluble T-cell activators in bioprocessing (2023)

Chimeric antigen receptor (CAR) T-cell therapy has emerged as a highly promising cell-based approach for the treatment of cancers. A key initial step in biomanufacturing is for T-cells to be activated before their transduction so as to promote their transfection and proliferation into high yields of CAR T-cells. Soluble activators provide an attractive alternative for biomanufacturing since they eliminate the cell losses when magnetic beads are removed, and this also simplifies bioprocessing. However, understanding the soluble activator exposures of human T-cells is a potential critical process parameter in the biomanufacturing of CAR T-cells. As alternative functional assays were assessed, it was found that upon activation, a major change in cell size was observed long before the cell numbers increased. Thus, a functional assay to measure the activity in culture supernates was developed based on measurements of T-cell diameters. Image analysis was used to measure the sizes of T-cells after 24, 48, or 72 hours of culture to determine the duration that most effectively distinguished variable activity levels. This activity assay was then used to determine the soluble activator levels over time, as well as to distinguish mechanisms of activity loss caused by binding to culture vessel surfaces, thermal degradation, or cell binding/internalization. The cell binding/internalization was by far the greatest contributor to activity decreases. Thus, this assay provides a means to monitor a key variable so as to increase the process understanding of CAR T-cell biomanufacturing. Additionally, the feasibility of refreezing many small T-cell aliquots with an acceptable recovery rate was demonstrated.

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Comparison of MIN6 cell aggregate formation methods and shear resistance (2019)

Islet transplantation has the potential to cure type 1 diabetes, thereby avoiding the need for a lifetime of daily insulin injections. However, to protect the islet transplant from the recipients host immune system currently requires lifelong immunosuppression. Alginate gel microencapsulation is one of the biological envelopes being developed as a physical barrier to block rejection by the recipient immune system. When using emulsification for cell encapsulation, aggregates of cells are exposed to high shear stresses that can impact their recovery. This study has investigated the aggregation of MIN6 cells to develop a model system for insulin producing beta-cells. Two aggregation methods were investigated, either using a shaking agitation system or a static multi-well system without shaking. MIN6 cell aggregates generated by both methods were analyzed for their recovery after exposure to shear stresses. A disaggregation method was introduced to examine the cellular viability of the component cells in aggregates and the cells remained viable within aggregates. Finally, the aggregates were encapsulated using 1.5% (w/v) or 5% (w/v) alginate. The 5% alginate yielded higher encapsulation efficiencies and a more spherical structure with a narrower size distribution of capsule diameters, and so should be the more suitable choice for the further development of large-scale encapsulation production processing.

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Computational modeling and performance characterization of an acoustic cell concentration and washing device (2019)

The success and clinical approval of CAR T-cell therapy has stimulated the development of manufacturing process technologies tailored for the needs of these new cell therapies. Cell concentration and washing is the most common unit operation such that it is crucial to perform this in a reliable and robust manner, with high yields. Acoustic cell separation provides a non-fouling filter alternative by trapping cells in a gentle ultrasonic standing wave. This innovative technology has the advantages of being operable in a closed system without cell losses to the much larger surfaces of conventional filters or in cylindrical centrifuges. However, the acoustic energy dissipation into heat causes temperature increases that need to be well understood and constrained to harmless ranges. Computational fluid dynamic models of the device energy sources and sinks were developed and investigated, including the effects of air cooling and different ultrasound power inputs. The internal temperature distributions showed that air cooling near the ultrasound source effectively cooled the device in the range acoustic power inputs were used. Thus, with the input power below 7 W at 3 mL/min flows, the temperature did not exceed 37˚C and so should not harm the cells. Within these constraints, design of experiment and modeling methods were used to maximize the separation yields as a function of the cell concentration, acoustic power and flow rate. This was applied to washing a freezing medium from Jurkat cells. A flow rate of 4 mL/min and input power of 3.8 W maximized the washed cell recovery at input cell concentration of 7x106 cell/mL, with the possibility of increasing the flow rate up to 7 mL/min without compromising the separation efficiency. This study highlights the applicability of this technology for cell separation and washing as well as the value of systematic engineering analysis to understand and maximize the performance of devices for cell therapy processing.

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Assessment of the Onset and Progress of Apoptosis via Raman Spectroscopy (2016)

Apoptotic and necrotic cell death is ultimately the cause of productivity loss in bioreactors used to produce therapeutic proteins. This study investigates the suitability of Raman spectroscopy to detect the onset and types of cell death in Chinese Hamster Ovary (CHO) cells - the most widely used cell type for therapeutic protein production. Apoptotic, necrotic or autophagic CHO cells producing tissue plasminogen activator were compared to uninduced cultures using Raman spectroscopy and principal component analysis (PCA). A fingerprint region was identified where several peaks change in the course of cell death. Further, uninduced cells were compared to cells sorted at different stages of apoptosis, in order to establish how early the onset of apoptosis could be detected. These results move past what has been described in literature, as we have shown that apoptosis-induced cells that score as viable in conventional apoptosis assays appear to have an altered biochemical composition compared to uninduced cells. Cells from different stages of fed-batch cultures were compared, and the results showed that Raman spectroscopy can be used to monitor the progress of a fed-batch culture. However, further work is required to elucidate the onset of apoptosis in fed-batch cultures. Future goals include assessing different inducers of apoptosis in order to construct a "library" of biochemical changes during apoptotic cell death, and developing automated classification models such as support vector machines to classify cell death types.

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Cell Death Dynamics Monitoring using Raman Micro-Spectroscopy (2015)

Biopharmaceuticals play a crucial role in curing diseases like Cancer and diabetes. Bioreactors are the heart of the industry. Cell losses due to cell death such as apoptosis and necrosis in the bioreactor decreases production efficiency and subsequently increases the cost of production. Furthermore, the study of apoptosis and necrosis cell death mechanisms has a great scientific and clinical importance in cancer therapy. In this project, Raman micro-spectroscopy is used to study apoptosis and necrosis in Chinese’s Hamster Ovary (CHO) cells that are one the main host cell lines used in the production of biopharmaceuticals. Apoptosis and Necrosis were induced in CHO cells using camptothecin and oxygen and glucose deprivation. The changes in the chemical composition of these enriched apoptotic and necrotic cell cultures were then analyzed using Raman spectroscopy which revealed novel biological concepts of the cell death process. Moreover, highly distinguishing Raman characteristics were identified for each death mode. These observations made by Raman spectroscopy were confirmed using a broad range of conventional and advanced biological assays in the field ranging from FACS analysis and fluorescent dyes to fluorescence microscopy. Studying Raman Spectra gave a clear image about DNA, RNA and Protein level changes during the process of apoptosis and necrosis in CHO cells. Using Principle Components Analysis (PCR) enabled viable, necrotic, early and late apoptotic populations to be clearly distinguished. This technology may provide the basis for the development of a non-invasive probe to monitor and predict cell death in bioreactor cultures in real-time and possibly allow cultures to avoid entering the cell death phase. In addition, the vast majority of cancer treatment methods involve cell death and apoptosis and, therefore, improving our knowledge about the biology of cell death will help support and advance research and treatment in this area.

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Effect of Amino Acid Limitation and Supplementation in Chinese Hamster Ovary Fed-batch Cultures (2015)

Fed-batch processes are the industrial norm for the production of recombinant proteins such as monoclonal antibodies (MAb) from Chinese hamster ovary (CHO) cells. Optimization of such processes is an important objective of industry process development groups. Amino acid availability is a key factor that is controlled to achieve the desired product yield and quality. In order to improve fed-batch productivity, the individual effects of limiting the three depleted amino acids were investigated for three antibody expressing CHO cell lines. Specifically, the effects of limiting glutamine, asparagine and cysteine on the cell growth, metabolism, antibody productivity and quality were investigated. Cysteine limitation was found to be detrimental to both the cell proliferation and the productivity for all three CHO cell lines. In contrast, asparagine limitation had no significant effect on either their growth or productivity. Glutamine limitation resulted in a reduction in growth but not in cell specific productivity, again for all three cell lines. Neither glutamine nor asparagine limitation significantly affected the MAb glycosylation. However, the fucosylation ratio was reduced in the absence of cysteine. It was confirmed that cysteine is a rate limiting factor for the productivity and growth of the three CHO cell lines. Replenishing cysteine after 1 day of the limitation allowed the cells to regain their growth and productivity; however, this was not observed after 2 days of cysteine limitation. Under cysteine limitation there was increased oxidative damage to the mitochondria, possibly caused by reduced synthesis of co-enzyme A which is essential for functionality of the TCA cycle. Finally, a fed-batch protocol was developed to improve the MAb productivity of CHO-DXB11 cells and the results were compared to the results with a commercial feed. Although use of the commercial feed resulted in higher maximum cell and final MAb concentrations, maintaining the levels of cysteine yielded cell specific production rates that were comparable to the commercial feed culture. Overall, the results of this study showed that amino acid limitations have varied effects on the performance of CHO cell cultures, such that it is important to focus process development efforts on the critical amino acids.

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Autophagy inhibition and hypertonicity increased monoclonal antibody production in Chinese hamster ovary fed-batch cultures (2013)

Biopharmaceuticals have improved the treatment of many diseases such as cancers and strokes, with much reduced adverse effects. However, high prices pose a major challenge to their widespread application. Improvements to cell culture processes can ease this burden, and facilitate the access to high quality biological medications for increasing sections of world society. This thesis addresses two methods to increase the productivity of the Chinese Hamster Ovary (CHO) cells expressing monoclonal antibodies using either autophagy inhibition or hypertonicity.Autophagy is a cellular process whereby intracellular components are degraded in response to nutrient limitations and other stresses. It has previously been shown that 3-methyladenine (3-MA) inhibition of autophagy can increase fed-batch tissue plasminogen activator production. This thesis investigated autophagy inhibition in fed-batch cultures of CHO cells producing monoclonal antibodies (MAb). Hypertonicity in mammalian cell cultures has also been shown to increase the MAb productivity of mammalian cell cultures. By investigating the timing and dose of 3-MA, more than two-fold increases in cell specific productivity and an up to two-fold increase in total MAb were obtained. Hypertonic conditions can similarly increase the productivity of fed-batch cultures by more than two-fold. The combination of these two approaches, however, did not result in any increase in productivity. Neither autophagy inhibition nor hypertonicity significantly changed the glycan profiles of the MAb product.

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