James Piret


Relevant Degree Programs

Affiliations to Research Centres, Institutes & Clusters


Open Research Positions

This list of possible research projects is non-exhaustive. It only shows positions that are specifically advertised in the G+PS website.

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - April 2022)
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 (2010 - 2021)
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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