PRISM :: Browsing by Author "Kallos, Michael S."

Browsing by Author "Kallos, Michael S."

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Open Access
Bioprocess Development for Large-Scale Production of Skin Derived Precursor Schwann Cells
(2018-08-16) Walsh, Tylor Douglas; Kallos, Michael S.; Schmidt, Tannin A.; Biernaskie, Jeff A.
Peripheral nerve and spinal cord injuries are debilitating, leading to lifelong complications and reduced quality of life. Cellular therapies have demonstrated beneficial outcomes when treating these injuries especially when using Schwann cells. However, there is currently no robust and reproducible method for producing Schwann cells at clinical scale. Bioprocesses that use bioreactors have significant advantages when scaling-up cellular therapies. Therefore, the research in this thesis was done to address this gap and develop methods, tools, and protocols to create a bioprocess for the large-scale expansion of Schwann cells. The hydrodynamics of the bioreactor were investigated by using CFD modeling, comparing velocity, shear rate, and energy dissipation rate at different agitation rates and their effect on cell expansion. The model that was generated can be used to scale up processes to larger, clinical and manufacturing scale, bioreactors. Upstream and downstream unit operations were then developed. Commercially available microcarriers were evaluated and tested in bioreactors to find the microcarrier that supported both inoculation and expansion of SKP-SCs. Different bioreactor platforms were evaluated, showing that controlling the process parameters increased cell densities. An in-depth DOE was conducted to find the best inoculation conditions, investigating which parameters had significant effects on cell attachment, distribution, and expansion. Detachment of SKP-SCs from microcarriers was investigated with different enzymes and agitation rates to develop an in-vessel passaging protocol that can easily be scale-up. After harvest, cryopreservation medium and cell density were investigated to ensure a quality product can be frozen and delivered to the patient. After the process was developed, 3 rat lines and 1 human line were tested. The process was reproducible and robust and easily adapted to human cells. Additional development is needed to use this process for nerve derived Schwann cells. This process was then integrated together and 150 x106 cells were produced from 3x106 in 7 days. Lastly, the expansion design space was investigated to determine the effects of pH, DO, and agitation on the expansion of SKP-SCs. All the tools and methods developed in this thesis can easily be adapted to nearly any bioprocess that utilize bioreactors for cellular therapies.
Open Access
Bioprocessing of human bone marrow mesecnchymal stem cells for the treatment of intervertebral disc degeneration
(2012) Yuan, Yifan; Kallos, Michael S.; Sen, Arindom
Lower back pain is a major medical problem in North America. From the biological point of view, the degeneration of intervertebral disc may be the main cause for lower back pain. The current study focused on using stem cell to differentiation into large number of healthy intervertebral disc cells. Human bone marrow mesenchymal stem cells (hBMMSCs) were used. This project has been divided into two parts: I. hBM-MSCs expansion, 2. hBM-MSCs differentiation. Experiments were carried out manipulating culture medium components ( oxygen tension, calcium, serum, pH) and agitation to improve the expansion of hBM-MSCs in bioreactors. After 33 days of culture under the developed protocol, approximately I 03 fold increase over the inoculation density had been obtained in suspension bioreactor culture. The differentiation of MSCs to nucleus pulposus-like cells was performed by using multiple growth factor cocktails and notochordal conditioned medium. There was higher expression of genes and proteins specific for nucleus pulposus cells, after exposure of MSCs to conditioned medium over 21 days than in basal medium.
Open Access
Cell cycle kinetics of expanding populations of mammalian neural stem cells
(2002) Alam, Sharmila; Kallos, Michael S.
Open Access
Characterization of cell mobility within neural stem cell aggregates
(2005) Wang, Tony Yi-Hsiang; Kallos, Michael S.
Open Access
Development of an extracellular matrix to improve pseudoislet survival for the treatment of Type I diabetes
(2019-09-12) Wong, Sarah; Ungrin, Mark D.; Kallos, Michael S.; Rancourt, Derrick E.
Throughout the islet transplantation process, many islets die due to removal from their native environment. Studies suggest that select extracellular matrices (ECMs) have the potential to support islet survival throughout transplantation. However, there is currently no overview evaluating ECM interactions with pseudoislets. In this study, three common methods of quantifying cell survival were applied to aggregates encapsulated in thin hydrogel sheets and their effectiveness was evaluated. Next, a Design of Experiments approach was used to evaluate composite hydrogel ECMs as a method of improving the survival of INS1 and primary human pseudoislets post-isolation. Encapsulating INS1 pseudoislets in [alginate] = 2wt/v%, [collagen I] ~ 4mg/mL, and [Matrigel] ~ 4mg/mL improved INS1 survival and proliferation when cultured for 3 days. Similarly, collagen I and Matrigel were found to have a neutral or positive effect on primary human pseudoislet survival when encapsulated in 2 wt/v% alginate gels and cultured for 5 days.
Open Access
Development of an injectable biomaterial scaffold for regeneration of the nucleus pulposus
(2011) Fisher, Stephanie; Kallos, Michael S.; Hunter, Christopher J.
Open Access
Development of bioprocesses for the stirred suspension bioreactor production of osteogenic and chondrogenic cells from embryonic stem cells
(2011) Alfred, Rosalind K.; Kallos, Michael S.
Open Access
Development of microbioreactors for embryonic stem cell bioprocess design
(2009) Millar, Vanessa Frances; Kallos, Michael S.
Open Access
Expansion of embryonic stem cells as aggregates in suspension culture bioreactors
(2006) Cormier, Jaymi T.; Kallos, Michael S.
Open Access
Gasification of Athabasca bitumen: hydrogen generation, kinetics, and in situ process design
(2012) Kapadia, Punitkumar Ramanlal; Gates, Ian D.; Kallos, Michael S.
Alberta, Canada has an immense amount of crude bitumen reserves in Athabasca, Cold Lake and Peace River deposits. Commercially, these reserves are exploited either by surface mining or in situ recovery methods in the form of liquid bitumen. Subsequently, the produced bitumen is upgraded to synthetic crude oil (SCO). SCO constitutes the main feed intake for conventional refineries wherein it is processed further to obtain fuel, petrochemicals, and lubricant products. Even though the current pathway of energy production from crude bitumen reserves is technically successful, commercially proven and economically viable, it requires large energy investments and high emission of pollutants to environment. Hence, there is a need for exploration of alternative energy vectors for production from these reserves. There are sufficient numbers of experimental and pilot scale studies demonstrating the possibility of hydrogen production from bitumen. Hydrogen is a clean fuel, has the highest energy content per unit mass, and is a required feedstock for the chemical and petrochemical industries. Given the extent of Athabasca oil sands reservoirs (over 1.7 trillion barrels); hydrogen generation from these resources would potentially have benefits to Alberta and Canada. The overall objective of the proposed research was to understand and optimize hydrogen generation by in situ gasification from bitumen reservoirs. The methods to enable this research were analysis of experimental pyrolysis, aquathermolysis, combustion data for Athabasca bitumen to obtain a reaction scheme and associated kinetic parameters, matching of combustion tube experiments to evaluate transport parameters, and simulation of field scale gasification recovery processes in Athabasca bitumen reservoir models. The key outcomes of the research were, • Evaluation of hydrogen generating potential from gasification of Athabasca bitumen, • Development of comprehensive reaction scheme to include pyrolysis, aquathermolysis, and combustion mechanisms for gasification of Athabasca bitumen, • Analyse of reaction zones in SAGD operation to provide estimates of acid gas production like hydrogen sulfide, carbon oxides and fuel gases like hydrogen, methane evolved during the SAGD field operation, and • In situ process design for bitumen gasification.
Open Access
Microscale Tissue Engineering and Contributors of the Cellular Niche
(2019-05-31) Al-Ani, Abdullah; Ungrin, Mark D.; Biernaskie, Jeff A.; Kallos, Michael S.; Rancourt, Derrick E.; Hirota, Simon Andrew; Gratzer, Paul F.
The behaviour of cells is modulated by their microenvironment or ‘niche’. While cellular therapies offer promising curative solutions for many diseases, the efficacy of transplanted cells is often hampered by a suboptimal microenvironment. One strategy to overcome this limitation is to reconstruct the niche of the cells of interest prior to transplantation. The central aim of this thesis is to develop novel tissue engineering approaches to further understand and reconstitute the cellular niche. While these approaches were specifically validated in the retinal and islet systems, they were also designed to be easily implemented in other biological systems. One project showcases a novel scaffold-free, scalable and injectable retinal pigment epithelium (RPE) microtissue for minimally-invasive transplantation. While RPE transplantation holds great potential to cure various retinal degenerative diseases, cells transplanted as cellular suspension exhibit suboptimal survival and function. Conversely, transplanting RPE as coherent cellular sheets has yielded better outcomes, but they are complex to transplant and produce at large scale. Our RPE microtissues were designed to capture the benefit of both approaches: namely, simplicity of production and transplantation, as well as enhanced performance. We found that our RPE microtissue exhibited superior cellular behaviour in terms of gene expression and in vitro function when compared to standard adherent culture. Another project presents a unique approach to produce transplants with a reconstituted cellular niche. This approach aims to repopulate the niche by incorporating finite amounts and proportions of niche cells into transplantable constructs. Using it enabled us to produce size-controlled pseudoislet constructs that contained various proportions of mesenchymal stem cells (MSCs), fibroblast and endothelial cells, and to quantitatively evaluate their in vitro performance. Further, applying this approach led us to discover more than one favourable condition that yielded improvements in islet cell performance in vitro. While the islets of Langerhans were used for biological validation, the approach was designed to be broadly applicable to various biological systems. In sum, this thesis offers several novel approaches for scientists to better understand and enhance the cellular niche. The simplicity, accessibility and scalability of these approaches render them suitable for both scientific applications and clinical translation.
Open Access
Mixing Rules for a NonCubic Equation of State
(2007) Kedge, Christopher J.; Kallos, Michael S.; Trebble, M. A.
Open Access
Modelling Stirred Suspension Bioreactors for Scalable Expansion of Pluripotent Stem Cells
(2018-06-25) Wyma, Alexander Arun; Kallos, Michael S.; De La Hoz Siegler, H.; Gates, Ian Donald; Edwards, William Brent; Kantzas, Apostolos
The emerging field of regenerative medicine seeks to leverage the potential of stem cell therapies to treat a wide variety of diseases and conditions. Sophisticated bioprocesses with engineering controls are required to produce the billions of cells required to facilitate these therapies. To this end, computational fluid dynamics (CFD) has been gaining popularity as a tool to characterize the hydrodynamic environment stem cells experience during bioprocessing. Recent studies have shown mammalian stem cell cultures have non-Newtonian viscosity, while CFD investigations of mammalian stem cell bioprocessing have assumed they are a Newtonian fluid. This study compares CFD simulations of 100mL and 500mL stirred suspension bioreactors with Newtonian and non-Newtonian viscosity models. Findings indicate shear stress, turbulent energy dissipation rate, power number, and turbulent power ratio are all significantly affected by the difference in viscosity models. Further, volume average turbulent energy dissipation rate from CFD studies with non-Newtonian viscosity was used to successfully maintain murine embryonic stem cell aggregate size distribution during bioreactor scale-up. The results of this study demonstrate the need for accurate representation of cell culture viscosity and highlight the power of CFD as a tool to control and optimize stem cell bioprocessing.
Open Access
Poly (ε-Caprolactone)-Silk Fibroin Based Functional Repair for Annulus Fibrosus Tears
(2019-01-24) Novin, Mana; Duncan, Neil A.; Kallos, Michael S.; Salo, Paul T.; Ungrin, Mark D.; Sen, Arindom; Lü, Qingye
Intervertebral disc degeneration with an associated bulged/herniated disc is a significant cause of low back pain. Annulus fibrosus (AF) tears and defects are a major clinical problem with no current treatments available for its closure and repair, resulting in risk of re-herniation. This thesis focuses on the chemical and mechanical characterization of a newly-designed biodegradable poly(ɛ-caprolactone)-silk fibroin (PCL-SF) as a potential candidate for the closure of irregular AF defects through minimally-invasive implantation. Thermoset PCL-SF scaffolds were produced with two concentrations of the PCL-diacrylate macromer solution (40% and 60% w/v) and five PCL:SF ratios (100:0, 90:10, 80:20, 70:30, and 60:40). Chemical characterization of the scaffolds confirmed the effective blending of PCL and SF macromolecules with uniform distribution of SF throughout the scaffolds and formation of β-sheet conformation in SF. Mechanical characterization of the scaffolds showed: (i) highly interconnected pores with pore sizes of 260–265 μm, (ii) tensile moduli and yield strains of 0.22-0.31 MPa and 41-61%, respectively, and (iii) compressive moduli of 0.11- 0.27 MPa. The above-mentioned porosities were within the range that reportedly supports AF cell penetration, adhesion, and accumulation of a collagen I rich extracellular matrix. The tensile moduli of tested scaffolds were in the range of human AF tissue in radial and axial directions. The compressive moduli were slightly less than native AF tissue but approximately an order of magnitude higher than those of other AF repair biomaterials. Additionally, the in vitro biodegradation rate of scaffolds was found to be slow enough to provide mechanical support in the time frame needed for AF regeneration. However, scaffolds were unable to exhibit shape-memory capabilities suitable for self-fitting in AF defects. Further optimization of the scaffold design with respect to shape memory capability for minimally-invasive delivery and self-fitting in AF defect will be required for clinical application.
Open Access
Process Development for the Expansion of Equine Cord Blood Mesenchymal Stem Cells in Stirred Suspension Bioreactors for the Treatment of Joint Injuries in Horses
(2018-04-30) Roberts, Erin L.; Kallos, Michael S.; Hart, David A.; De La Hoz Siegler, H.
Musculoskeletal injuries are the leading cause of lameness and loss of performance in horses with conventional treatments having high rates of re-injury. Equine Mesenchymal stem cells (eMSCs), specifically equine cord blood derived MSCs (eCB-MSCs) are a promising treatment alternative owing to their ability to differentiate into cartilage, as well as due to their immunomodulating properties. However, treatments can require cell numbers in the millions or billions, therefore conventional expansion methods using static T-flasks are typically inadequate in achieving these cell numbers. Expansion of eCB-MSCs in stirred suspension bioreactors with microcarriers as an attachment surface has the potential to obtain a clinically relevant number of cells while decreasing cost, time and labour requirements and increasing reproducibility and yield when compared to static expansion. As eCB-MSCs had not yet been expanded in stirred suspension bioreactors, a robust protocol was required to expand these cells using this method. This study outlines the process development of expansion, detailing the inoculation phase, the expansion phase, and the harvesting phase, followed by further testing on different donor cells. The process achieved maximum cell densities up to 75,000 cells/cm2 corresponding to 40 million cells in a 100mL bioreactor, with a harvesting efficiency of up to 80%, corresponding to a yield of 32 million cells in a 100mL bioreactor. Further testing is required to ensure eCB-MSCs maintained phenotype and functionality, however this study lays the groundwork for the large scale bioprocess of the expansion of eCB-MSCs.
Open Access
Prototype design and temperature uniformity on loop-mediated isothermal amplification temperature control system
(2012) Cao, Rui; Kallos, Michael S.
Malaria diagnostics needs to be rapid, accurate and cost effective to ensure proper treatment is received. Loop-mediated isothermal amplification (LAMP) DNA amplification method has these characteristics. This nucleic acid amplification test (NAT) can identify parasite genus and species accurately and even detect drug resistance. Compared to conventional microscopy, which is the main tool used for detection and identification in the field, the LAMP method has significantly higher sensitivity and specificity. However, the problem with LAMP is the cost of the reagents, and the cost and operation of the equipment to run (heating blocks/thermal cyders) and reliably read out the assays (spectrophotometers). This dissertation discusses about low-cost prototype design of heating block and temperature uniformity control. For the prototype design, Chapter IV introduces the constituents of hardware system first and then introduces the design of heating and cooling system based on Thermoelectric Cooler (TEC) and finally introduces two assistant departments-heated lid and fan briefly. For the research on heating block temperature uniformity, Chapter V includes establishing the finite element simulation model of the sample block to obtain the temperature field. From the result of the simulation, the exiting model already has good temperature uniformity. Further work would include testing the model experimentally. Finally, Chapter VI includes research on marketing analysis for our device in developing countries.
Open Access
Robust bioprocess design and evaluation of commercial media for the serial expansion of human induced pluripotent stem cell aggregate cultures in vertical-wheel bioreactors
(2024-07-29) Borys, Breanna S.; Dang, Tiffany; Worden, Hannah; Larijani, Leila; Corpuz, Jessica M.; Abraham, Brett D.; Gysel, Emilie J.; Malinovska, Julia; Krawetz, Roman; Revay, Tamas; Argiropoulos, Bob; Rancourt, Derrick E.; Kallos, Michael S.; Jung, Sunghoon
Abstract Background While pluripotent stem cell (PSC) therapies move toward clinical and commercial applications at a rapid rate, manufacturing reproducibility and robustness are notable bottlenecks in regulatory approval. Therapeutic applications of PSCs require large cell quantities to be generated under highly robust, well-defined, and economically viable conditions. Small-scale and short-term process optimization, however, is often performed in a linear fashion that does not account for time needed to verify the bioprocess protocols and analysis methods used. Design of a reproducible and robust bioprocess should be dynamic and include a continuous effort to understand how the process will respond over time and to different stresses before transitioning into large-scale production where stresses will be amplified. Methods This study utilizes a baseline protocol, developed for the short-term culture of PSC aggregates in Vertical-Wheel® bioreactors, to evaluate key process attributes through long-term (serial passage) suspension culture. This was done to access overall process robustness when performed with various commercially available media and cell lines. Process output variables including growth kinetics, aggregate morphology, harvest efficiency, genomic stability, and functional pluripotency were assessed through short and long-term culture. Results The robust nature of the expansion protocol was demonstrated over a six-day culture period where spherical aggregate formation and expansion were observed with high-fold expansions for all five commercial media tested. Profound differences in cell growth and quality were revealed only through long-term serial expansion and in-vessel dissociation operations. Some commercial media formulations tested demonstrated maintenance of cell growth rates, aggregate morphology, and high harvest recovery efficiencies through three bioreactor serial passages using multiple PSC lines. Exceptional bioprocess robustness was even demonstrated with sustained growth and quality maintenance over 10 serial bioreactor passages. However, some commercial media tested proved less equipped for serial passage cultures in bioreactors as cultures led to cell lysis during dissociation, reduction in growth rates, and a loss of aggregate morphology. Conclusions This study demonstrates the importance of systematic selection and testing of bioprocess input variables, with multiple bioprocess output variables through serial passages to create a truly reproducible and robust protocol for clinical and commercial PSC production using scalable bioreactor systems.
Open Access
Scaling Cellular Aggregate Production in a Microwell System
(2020-09-14) Kondro, Douglas A.; Ungrin, Mark D.; Kallos, Michael S.; Nezhad, Amir S.
Microtissues, in the form of cellular aggregates, offer a promising format for cell-based therapies, such as the treatment of type 1 diabetes with beta cell replacement. One major challenge is producing a sufficient quantity of microtissue for clinical use. For instance, type 1 diabetes requires approximately 1 million cellular aggregates for treatment of a single individual. Static culture methods of producing aggregates, such as the microwell system, offer an accessible format of producing high quality, size-controlled cellular aggregates, however they cannot easily generate the quantity required for a clinical treatment. This thesis scales an existing microwell format by producing a robust, self-contained, multilayered microwell bioreactor capable of generating over 250,000 cellular aggregates in a well plate footprint. The design of the microwell bioreactor consists of a sealed chamber where cells aggregate on thin sheets of microwells in the device. The microwells are made with an oxygen permeable polymer to facilitate gas exchange in the sealed system, that was demonstrated to deliver approximately 4 times the oxygen to cells in comparison with the well plate format. The design process utilizes hot-embossing, allowing for efficient and scalable manufacturing, with a demonstrated cycle time as low as 15 seconds per microwell sheet. The final device is robust, capable of withstanding centrifugation up to 2000 g, and autoclaving sterilization. Further, the protocol for cell culture is simplified as it allows for removal of >97% of media during a media change without disturbing aggregates, and removal of >98% of cellular aggregates after long term culturing. Biological validation and demonstration of clinical utility was performed using 12 donor islet batches. The microwell bioreactor could scale the production of functional pseudoislets in the form of size-controlled cellular aggregates. Pseudoislets were demonstrated to have indistinguishable functional performance in compared to previously published methods. Applicability of this novel system was also demonstrated for other diseases by successfully differentiating pluripotent stem cells and aggregating testicular organoids. The unique design of the microwell bioreactor provides a straightforward tool to enable researchers to immediately scale static culture experiments, allowing for translation of cell-based therapies to clinical use.
Open Access
Scaling up neural stem cell expansion in suspension bioreactors
(2005) Gilbertson, Jane Anne; Kallos, Michael S.
Open Access
Small-scale bioprocessing embryonic stem cells
(2011) Van Winkle, Allison Patricia; Kallos, Michael S.; Gates, Ian D.
Embryonic stem cells are capable of self-renewal and can be driven to differentiate into cell types from all three germ layers making them a source of cells with tremendous potential in the treatment of debilitating diseases and injuries. Expanding and differentiating cells in suspension bioreactors is a critical step towards widespread use of stem cells in medicine. The goal of this project was to develop a small-scale microbioreactor array for high throughput experimentation in order to improve the protocols used for expansion and differentiation of populations of embryonic stem cells. Initial experiments were performed with a small prototype array with the aim of replicating the cell expansion obtained in standard 100 mL bioreactors. These results of these experiments led to the hypothesis that the fluid environment created in the bioreactors was substantially different between these two scales. A finite element simulation was created to compare the hydrodynamic environments. Following this, a numerical simulation was used to study the effect of diffusion on embryonic stem cell differentiation in aggregate form. These simulation studies have important implications for future bioprocess design.