James Piret

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

View record

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.

View record

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.

View record

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.

View record

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.

View record

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.

View record

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.

View record

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.

View record

No abstract available.

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.

View record

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.

View record