Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (N... more Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (NSC) have been determined. We examined suspension culture bioreactors of murine NSC, and concluded that the oxygen level should be kept high (20%), and the osmolarity of the medium should be kept low (below 400 mOsm/kg). The pH of the medium was found to have a large effect on cell proliferation, and the best growth characteristics were obtained within an optimum pH range of 7. 1 to 7.5. The inoculation conditions were also seen to have a large effect not only on the growth characteristics, but also on the number of cells that die in the initial stages of the culture. For large expansion of cells, low inoculum levels (10(4) cells/mL) and single-cell suspensions proved superior, whereas, for fast expansion of cells, higher inoculum levels (10(5) cells/mL) and spheroid inoculum forms were preferred. The inoculum temperature of the medium did not have a large effect on growth characteristics, but the pH greatly influenced cell proliferation. Inoculum pH levels should also be kept between 7.1 and 7.5. If these protocols are followed, high multiplication ratios and viabilities can be obtained in a 5-day batch suspension culture bioreactor run. A large number of cells could then be used in animal models for testing of neural drugs and in research and development toward cures for neurodegenerative disorders such as multiple sclerosis (MS) and Huntington's and Parkinson's disease. The results presented here also point the way toward studies on in vitro expansion of human neural stem cells.
Neural stem cells (NSCs) are primitive cells that are the "parent" cells of all... more Neural stem cells (NSCs) are primitive cells that are the "parent" cells of all the cells in the central nervous system (CNS). Their discovery in 1992 opened the door to a multitude of potential therapies and treatments to cure neurodegenerative diseases such as Parkinson's disease, multiple sclerosis, and Huntington's disease, which affect millions of people worldwide and cost billions of dollars in health care each year. This study proposes optimal serial passaging protocols so that mammalian neural stem cells can effectively be grown in suspension culture. We examined stationary culture passaging protocols and developed our own optimal procedure. Also examined was the effect of serially cultivating the neural stem cells in suspension culture for an extended period of time. The cells were grown for over 35 days in suspension with an overall multiplication ratio of over 10(7) with no decrease in growth rate, maximum cell density, or viability. The cells also remained karyotypically normal through 25 doublings and retained their ability to be differentiated into all the major cell types of the CNS-neurons, astrocytes, and oligodendrocytes. For the first time, mammalian neural stem cells were grown on a larger scale in suspension culture and maintained their stem cell characteristics. A semicontinuous scheme for large-scale production is also presented.
Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for r... more Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for regenerating tissues lost to injuries and degenerative diseases. For clinical implementation of this technology, a quality controlled, reproducible culture system is necessary for the expansion and differentiation of the cells. Used in many bioprocess applications, suspension bioreactors have gained considerable attention for the regulated large-scale expansion of cells. The current study presents a bioreactor process for the large-scale expansion of undifferentiated murine ESCs as aggregates. In this system, the level of ESC aggregation and differentiation was effectively controlled by adjusting shear forces and inoculation density, achieving a 31-fold expansion in 5 days. Pluripotency markers Oct-4, Nanog, SSEA-1, ALP, and rex-1 were assessed using flow cytometry analysis and gene expression profiles and showed that the undifferentiated nature of the cells within the ESC aggregates was maintained. Colony-forming efficiencies and embryoid body formation tests of the expanded cultures demonstrated that characteristic functional attributes of undifferentiated cells were not lost. Overcoming a major impediment in the area of ESC expansion, this study describes a successful process for the controlled and reproducible largescale expansion of ESCs using suspension culture bioreactors.
Multipotent neural precursors can be cultured in suspension bioreactors as aggregates of stem cel... more Multipotent neural precursors can be cultured in suspension bioreactors as aggregates of stem cells and progenitor cells. However, it is important to limit the size of the aggregates, as necrotic centers may develop at very large diameters. Previously, we have shown that the hydrodynamics within a suspension bioreactor can be used to control the diameter of NSC aggregates (DMAVG<150 μm) below sizes where necrosis would be expected to occur. In the present study, power law correlations were developed for our bioreactors showing the dependence of the maximum mean aggregate diameter on both the kinematic viscosity of the medium and the power input per unit mass of medium. The power input was manipulated by changing the agitation rate (60–100 rpm), and the viscosity was manipulated through the addition of non-toxic levels of carboxymethylcellulose. The study also confirmed that the maximum liquid shear generated at the surface of the aggregates was sufficient to dislodge single cells, thus limiting the maximum diameter of the aggregates, without causing cell damage (τmax=9.76 dyn/cm2). This is a first step in the development of a reproducible, scaled-up process for the production of neural stem cells for therapeutic applications including the treatment of neurodegenerative disorders and acute central nervous system injuries.
Industrial & Engineering Chemistry Research, Jan 1, 2001
Mammalian neural stem cells hold great promise for the treatment of central nervous system disord... more Mammalian neural stem cells hold great promise for the treatment of central nervous system disorders. However, to be a viable clinical treatment for the millions of individuals afflicted with these disorders, it is necessary to develop cell expansion protocols. Although difficult to grow in bioreactors, neural stem cells can be expanded in carefully designed media as aggregates of brain tissue. The objective of this study was to examine the control of the aggregate size in a batch culture by manipulating the agitation rate, and hence the liquid ...
Medical & Biological Engineering & Computing, Jan 1, 2003
A relatively new approach to the treatment of neurodegenerative diseases is the direct use of neu... more A relatively new approach to the treatment of neurodegenerative diseases is the direct use of neural stem cells (NSCs) as therapeutic agents. The expected demand for treatment from the millions of afflicted individuals, coupled with the expected demand from biotechnology companies creating therapies, has fuelled the need to develop large-scale culture methods for these cells. The rapid pace of discovery in this area has been assisted through the use of animal model systems, enabling many experiments to be performed quickly and effectively. This review focuses on recent developments in expanding human and murine NSCs on a large scale, including the development of new serum-free media and bioreactor protocols. In particular, engineering studies that characterise important scale-up parameters are examined, including studies examining the effects of long-term culture of NSCs in suspension bioreactors. In addition, recent advances in the human NSC system are reviewed, including techniques for the evaluation of NSC characteristics.
Mammalian neural stem cells (NSC) offer great promise as therapeutic agents for the treatment of ... more Mammalian neural stem cells (NSC) offer great promise as therapeutic agents for the treatment of central nervous system disorders. As a consequence of the large numbers of cells that will be needed for drug testing and transplantation studies, it is necessary to develop protocols for the large-scale expansion of mammalian NSC. Neural stem cells and early progenitor cells can be expanded in vitro as aggregates in controlled bioreactors using carefully designed media. The first objective of this study was to determine if it is possible to maintain a population of murine neural stem and progenitor cells as aggregates in suspension culture bioreactors over extended periods of time. We discovered that serial passaging of a mixture of aggregates sizes resulted in high viabilities, high viable cell densities, and good control of aggregate diameter. When the NSC aggregates were serially subcultured three times without mechanical dissociation, a total multiplication ratio of 2.9 × 103 was achieved over a period of 12 days, whereas the aggregate size was controlled (mean diameter less than 150 μm) below levels at which necrosis would occur. Moreover, cell densities of 1.0 × 106 cells/mL were repeatedly achieved in batch culture with viabilities exceeding 80%. The second objective was to examine the proliferative potential of single cells shed from the surface of these aggregates. We found that the single cells, when subcultured, retained the capacity to generate new aggregates, gave rise to cultures with high viable cell densities and were able to differentiate into all of the primary cell phenotypes in the central nervous system.
The successful dissociation of mammalian neural stem cell (NSC) aggregates (neurospheres) into a ... more The successful dissociation of mammalian neural stem cell (NSC) aggregates (neurospheres) into a single-cell suspension is an important procedure when expanding NSCs for clinical use, or when performing important assays such as clonal analyses. Until now, researchers have had to rely primarily on destructive mechanical methods such as trituration with a pipette tip to break apart the aggregates. In this study we report on a new chemical dissociation procedure that is efficient, cost effective, reproducible, and much less harmful to murine NSCs than both mechanical and enzymatic techniques. This method, involving the manipulation of environmental pH levels, resulted in 40% higher measured cell densities and 15-20% higher viabilities compared with mechanical dissociation. Moreover, chemical dissociation resulted in the production of significantly less cellular debris. Chemical dissociation was found to have no adverse effects on the long-term proliferation of the NSCs, which retained the ability to proliferate, form neurospheres, self-renew, and exhibit multipotentiality. This chemical method represents a new approach for the dissociation of tissues.
Increasing attention has been drawn towards pluripotent embryonic stem cells (ESCs) and their pot... more Increasing attention has been drawn towards pluripotent embryonic stem cells (ESCs) and their potential use as the primary material in various tissue engineering applications. Successful clinical implementation of this technology would require a quality controlled reproducible culture system for the expansion of the cells to be used in the generation of functional tissues. Recently, we showed that suspension bioreactors could be used in the regulated large-scale expansion of highly pluripotent murine ESCs. The current study illustrates that these bioreactor protocols can be adapted for long term culture and that murine ESC cultures remain highly undifferentiated, when serially passaged in suspension bioreactors for extended periods. Flow cytometry analysis and gene expression profiles of several pluripotency markers, in addition to colony and embryoid body (EB) formation tests were conducted at the start and end of the experiment and all showed that the ESC cultures remained highly undifferentiated over extended culture time in suspension. In vivo teratoma formation and in vitro differentiation into neural, cardiomyocyte, osteoblast and chondrocyte lineages, performed at the end of the long term culture, further supported the presence of functional and undifferentiated ESCs in the expanded population. Overall, this system enables the controlled expansion of highly pluripotent murine ESC populations.
Since the derivation of human embryonic stem (hES) cells, their translation to clinical therapies... more Since the derivation of human embryonic stem (hES) cells, their translation to clinical therapies has been met with several challenges, including the need for large-scale expansion and controlled differentiation processes. Suspension bioreactors are an effective alternative to static culture flasks as they enable the generation of clinically relevant cell numbers with greater efficacy in a controlled culture system. We, along with other groups, have developed bioreactor protocols for the expansion of pluripotent murine ES cells. Here we present a novel bioreactor protocol that yields a 25-fold expansion of hES cells over 6 days. Using immunofluorescence, flow cytometry, and teratoma formation assays, we demonstrated that these bioreactor cultures retained high levels of pluripotency and a normal karyotype. Importantly, the use of bioreactors enables the expansion of hES cells in the absence of feeder layers or matrices, which will facilitate the adaptation of good manufacturing process (GMP) standards to the development of hES cell therapies.
Mutations in the pathways regulating mammary epithelial stem cell (MESC) self-renewal and differe... more Mutations in the pathways regulating mammary epithelial stem cell (MESC) self-renewal and differentiation are currently hypothesized to result in uncontrolled cell division and, in turn, breast tumor formation. Although research is aggressively being pursued to understand how such pathways result in breast cancer formation, current studies have been greatly limited by MESC scarcity. To address this issue, this study has successfully developed large-scale expansion protocols for MESC through the subculture of murine mammary epithelial tissue aggregates, called mammospheres, in suspension bioreactors. Growth kinetics of mammospheres cultured in 125 mL suspension bioreactors and T-flasks were found to be comparable, achieving cell densities of 3.10 × 105 and 2.75 × 105 cells/mL, respectively. This corresponded to a 4-fold expansion over 8 days. Yields were also found to be strongly affected by liquid shear forces, where high agitation rates reduced overall cell numbers. Bioreactor cultures were scaled up to 1000 mL operating volumes, resulting in the production of 4.21 × 108 total cells (5.6-fold expansion) from a single passage. Furthermore, intermittent replacement of culture medium with fresh medium dramatically improved maximum cell densities, resulting in an 11-fold expansion, thereby enabling the generation of stem cells in quantities sufficient for standard biochemical and genetic analyses. After being cultured in suspension bioreactors for several passages, analysis by flow cytometry of Ki-67 revealed that 85% of the population was composed of proliferating cells. The successful development of expansion protocols for MESC aggregates in suspension bioreactors makes available experimental avenues that were not previously accessible for breast cancer research, thereby facilitating future investigations into elucidating the role of MESCs in breast cancer tumorigenesis.
Large-scale expansion of neural stem and progenitor cells will be essential for clinically treati... more Large-scale expansion of neural stem and progenitor cells will be essential for clinically treating the large number of patients suffering from neurodegenerative disorders such as Parkinson's disease. Other applications of neural stem cell technology include further research in areas such as neural development or drug testing. Neural stem cells can be grown in vitro as tissue aggregates known as neurospheres, and in the current study, experiments were performed to determine the spatial arrangement and behavior of the cells within the neurosphere structure. A protocol utilizing sulfonated lipophilic fluorescent dyes was developed to effectively label populations of neural stem and progenitor cells without compromising cell density during culture. Cells retained the labels for at least 7 days. Using the labeling protocol, we discovered that the cells within the neurospheres were mobile and, moreover, the cells on the periphery of the neurospheres could migrate into the center of the neurospheres. Most important, the mixing time of two merging neurospheres was observed to be the same order of magnitude as the neural stem cell doubling time (∼20 h). This study is the first to show that the neurosphere system is dynamic, and these results will serve as a stepping stone to more in-depth studies of the neurosphere microenvironment.
Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for r... more Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for regenerating tissues lost to injuries and degenerative diseases. For clinical implementation of this technology, a quality controlled, reproducible culture system is necessary for the expansion and differentiation of the cells. Used in many bioprocess applications, suspension bioreactors have gained considerable attention for the regulated large-scale expansion of cells. The current study presents a bioreactor process for the large-scale expansion of undifferentiated murine ESCs as aggregates. In this system, the level of ESC aggregation and differentiation was effectively controlled by adjusting shear forces and inoculation density, achieving a 31-fold expansion in 5 days. Pluripotency markers Oct-4, Nanog, SSEA-1, ALP, and rex-1 were assessed using flow cytometry analysis and gene expression profiles and showed that the undifferentiated nature of the cells within the ESC aggregates was maintained. Colony-forming efficiencies and embryoid body formation tests of the expanded cultures demonstrated that characteristic functional attributes of undifferentiated cells were not lost. Overcoming a major impediment in the area of ESC expansion, this study describes a successful process for the controlled and reproducible largescale expansion of ESCs using suspension culture bioreactors.
The aim of this study was to develop a scaleable process to expand pancreatic endocrine tissue (i... more The aim of this study was to develop a scaleable process to expand pancreatic endocrine tissue (i.e., aggregates or islet-like structures) in suspension bioreactors. Key issues addressed included (i) serum-free media, (ii) cell inoculation density, (iii) medium pH, and (iv) aggregate dissociation. Suspension bioreactors were inoculated with pancreatic neonatal tissue and operated under controlled conditions for a 9-day period. Medium studies showed that a new serum-free medium developed in our laboratory was capable of supporting endocrine cell expansion. An inoculation density of 127,000 cells/mL resulted in more than a 7.5-fold increase in the number of insulin-positive cells after 9 days. The resulting population consisted of single cells and many islet-like aggregates that contained all of the endocrine cell types (including insulin-positive, glucagon-positive, somatostatin-positive, and pancreatic polypeptide-positive cells). Furthermore, the cell aggregates exhibited a glucose-responsive behavior. This study represents a significant milestone on the path to the effective expansion of human islet-like tissue in bioreactors that may be used for cell therapy to treat Type 1 diabetes.
Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (N... more Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (NSC) have been determined. We examined suspension culture bioreactors of murine NSC, and concluded that the oxygen level should be kept high (20%), and the osmolarity of the medium should be kept low (below 400 mOsm/kg). The pH of the medium was found to have a large effect on cell proliferation, and the best growth characteristics were obtained within an optimum pH range of 7. 1 to 7.5. The inoculation conditions were also seen to have a large effect not only on the growth characteristics, but also on the number of cells that die in the initial stages of the culture. For large expansion of cells, low inoculum levels (10(4) cells/mL) and single-cell suspensions proved superior, whereas, for fast expansion of cells, higher inoculum levels (10(5) cells/mL) and spheroid inoculum forms were preferred. The inoculum temperature of the medium did not have a large effect on growth characteristics, but the pH greatly influenced cell proliferation. Inoculum pH levels should also be kept between 7.1 and 7.5. If these protocols are followed, high multiplication ratios and viabilities can be obtained in a 5-day batch suspension culture bioreactor run. A large number of cells could then be used in animal models for testing of neural drugs and in research and development toward cures for neurodegenerative disorders such as multiple sclerosis (MS) and Huntington&#39;s and Parkinson&#39;s disease. The results presented here also point the way toward studies on in vitro expansion of human neural stem cells.
Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (N... more Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (NSC) have been determined. We examined suspension culture bioreactors of murine NSC, and concluded that the oxygen level should be kept high (20%), and the osmolarity of the medium should be kept low (below 400 mOsm/kg). The pH of the medium was found to have a large effect on cell proliferation, and the best growth characteristics were obtained within an optimum pH range of 7. 1 to 7.5. The inoculation conditions were also seen to have a large effect not only on the growth characteristics, but also on the number of cells that die in the initial stages of the culture. For large expansion of cells, low inoculum levels (10(4) cells/mL) and single-cell suspensions proved superior, whereas, for fast expansion of cells, higher inoculum levels (10(5) cells/mL) and spheroid inoculum forms were preferred. The inoculum temperature of the medium did not have a large effect on growth characteristics, but the pH greatly influenced cell proliferation. Inoculum pH levels should also be kept between 7.1 and 7.5. If these protocols are followed, high multiplication ratios and viabilities can be obtained in a 5-day batch suspension culture bioreactor run. A large number of cells could then be used in animal models for testing of neural drugs and in research and development toward cures for neurodegenerative disorders such as multiple sclerosis (MS) and Huntington&#39;s and Parkinson&#39;s disease. The results presented here also point the way toward studies on in vitro expansion of human neural stem cells.
Neural stem cells (NSCs) are primitive cells that are the &quot;parent&quot; cells of all... more Neural stem cells (NSCs) are primitive cells that are the &quot;parent&quot; cells of all the cells in the central nervous system (CNS). Their discovery in 1992 opened the door to a multitude of potential therapies and treatments to cure neurodegenerative diseases such as Parkinson&#39;s disease, multiple sclerosis, and Huntington&#39;s disease, which affect millions of people worldwide and cost billions of dollars in health care each year. This study proposes optimal serial passaging protocols so that mammalian neural stem cells can effectively be grown in suspension culture. We examined stationary culture passaging protocols and developed our own optimal procedure. Also examined was the effect of serially cultivating the neural stem cells in suspension culture for an extended period of time. The cells were grown for over 35 days in suspension with an overall multiplication ratio of over 10(7) with no decrease in growth rate, maximum cell density, or viability. The cells also remained karyotypically normal through 25 doublings and retained their ability to be differentiated into all the major cell types of the CNS-neurons, astrocytes, and oligodendrocytes. For the first time, mammalian neural stem cells were grown on a larger scale in suspension culture and maintained their stem cell characteristics. A semicontinuous scheme for large-scale production is also presented.
Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for r... more Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for regenerating tissues lost to injuries and degenerative diseases. For clinical implementation of this technology, a quality controlled, reproducible culture system is necessary for the expansion and differentiation of the cells. Used in many bioprocess applications, suspension bioreactors have gained considerable attention for the regulated large-scale expansion of cells. The current study presents a bioreactor process for the large-scale expansion of undifferentiated murine ESCs as aggregates. In this system, the level of ESC aggregation and differentiation was effectively controlled by adjusting shear forces and inoculation density, achieving a 31-fold expansion in 5 days. Pluripotency markers Oct-4, Nanog, SSEA-1, ALP, and rex-1 were assessed using flow cytometry analysis and gene expression profiles and showed that the undifferentiated nature of the cells within the ESC aggregates was maintained. Colony-forming efficiencies and embryoid body formation tests of the expanded cultures demonstrated that characteristic functional attributes of undifferentiated cells were not lost. Overcoming a major impediment in the area of ESC expansion, this study describes a successful process for the controlled and reproducible largescale expansion of ESCs using suspension culture bioreactors.
Multipotent neural precursors can be cultured in suspension bioreactors as aggregates of stem cel... more Multipotent neural precursors can be cultured in suspension bioreactors as aggregates of stem cells and progenitor cells. However, it is important to limit the size of the aggregates, as necrotic centers may develop at very large diameters. Previously, we have shown that the hydrodynamics within a suspension bioreactor can be used to control the diameter of NSC aggregates (DMAVG<150 μm) below sizes where necrosis would be expected to occur. In the present study, power law correlations were developed for our bioreactors showing the dependence of the maximum mean aggregate diameter on both the kinematic viscosity of the medium and the power input per unit mass of medium. The power input was manipulated by changing the agitation rate (60–100 rpm), and the viscosity was manipulated through the addition of non-toxic levels of carboxymethylcellulose. The study also confirmed that the maximum liquid shear generated at the surface of the aggregates was sufficient to dislodge single cells, thus limiting the maximum diameter of the aggregates, without causing cell damage (τmax=9.76 dyn/cm2). This is a first step in the development of a reproducible, scaled-up process for the production of neural stem cells for therapeutic applications including the treatment of neurodegenerative disorders and acute central nervous system injuries.
Industrial & Engineering Chemistry Research, Jan 1, 2001
Mammalian neural stem cells hold great promise for the treatment of central nervous system disord... more Mammalian neural stem cells hold great promise for the treatment of central nervous system disorders. However, to be a viable clinical treatment for the millions of individuals afflicted with these disorders, it is necessary to develop cell expansion protocols. Although difficult to grow in bioreactors, neural stem cells can be expanded in carefully designed media as aggregates of brain tissue. The objective of this study was to examine the control of the aggregate size in a batch culture by manipulating the agitation rate, and hence the liquid ...
Medical & Biological Engineering & Computing, Jan 1, 2003
A relatively new approach to the treatment of neurodegenerative diseases is the direct use of neu... more A relatively new approach to the treatment of neurodegenerative diseases is the direct use of neural stem cells (NSCs) as therapeutic agents. The expected demand for treatment from the millions of afflicted individuals, coupled with the expected demand from biotechnology companies creating therapies, has fuelled the need to develop large-scale culture methods for these cells. The rapid pace of discovery in this area has been assisted through the use of animal model systems, enabling many experiments to be performed quickly and effectively. This review focuses on recent developments in expanding human and murine NSCs on a large scale, including the development of new serum-free media and bioreactor protocols. In particular, engineering studies that characterise important scale-up parameters are examined, including studies examining the effects of long-term culture of NSCs in suspension bioreactors. In addition, recent advances in the human NSC system are reviewed, including techniques for the evaluation of NSC characteristics.
Mammalian neural stem cells (NSC) offer great promise as therapeutic agents for the treatment of ... more Mammalian neural stem cells (NSC) offer great promise as therapeutic agents for the treatment of central nervous system disorders. As a consequence of the large numbers of cells that will be needed for drug testing and transplantation studies, it is necessary to develop protocols for the large-scale expansion of mammalian NSC. Neural stem cells and early progenitor cells can be expanded in vitro as aggregates in controlled bioreactors using carefully designed media. The first objective of this study was to determine if it is possible to maintain a population of murine neural stem and progenitor cells as aggregates in suspension culture bioreactors over extended periods of time. We discovered that serial passaging of a mixture of aggregates sizes resulted in high viabilities, high viable cell densities, and good control of aggregate diameter. When the NSC aggregates were serially subcultured three times without mechanical dissociation, a total multiplication ratio of 2.9 × 103 was achieved over a period of 12 days, whereas the aggregate size was controlled (mean diameter less than 150 μm) below levels at which necrosis would occur. Moreover, cell densities of 1.0 × 106 cells/mL were repeatedly achieved in batch culture with viabilities exceeding 80%. The second objective was to examine the proliferative potential of single cells shed from the surface of these aggregates. We found that the single cells, when subcultured, retained the capacity to generate new aggregates, gave rise to cultures with high viable cell densities and were able to differentiate into all of the primary cell phenotypes in the central nervous system.
The successful dissociation of mammalian neural stem cell (NSC) aggregates (neurospheres) into a ... more The successful dissociation of mammalian neural stem cell (NSC) aggregates (neurospheres) into a single-cell suspension is an important procedure when expanding NSCs for clinical use, or when performing important assays such as clonal analyses. Until now, researchers have had to rely primarily on destructive mechanical methods such as trituration with a pipette tip to break apart the aggregates. In this study we report on a new chemical dissociation procedure that is efficient, cost effective, reproducible, and much less harmful to murine NSCs than both mechanical and enzymatic techniques. This method, involving the manipulation of environmental pH levels, resulted in 40% higher measured cell densities and 15-20% higher viabilities compared with mechanical dissociation. Moreover, chemical dissociation resulted in the production of significantly less cellular debris. Chemical dissociation was found to have no adverse effects on the long-term proliferation of the NSCs, which retained the ability to proliferate, form neurospheres, self-renew, and exhibit multipotentiality. This chemical method represents a new approach for the dissociation of tissues.
Increasing attention has been drawn towards pluripotent embryonic stem cells (ESCs) and their pot... more Increasing attention has been drawn towards pluripotent embryonic stem cells (ESCs) and their potential use as the primary material in various tissue engineering applications. Successful clinical implementation of this technology would require a quality controlled reproducible culture system for the expansion of the cells to be used in the generation of functional tissues. Recently, we showed that suspension bioreactors could be used in the regulated large-scale expansion of highly pluripotent murine ESCs. The current study illustrates that these bioreactor protocols can be adapted for long term culture and that murine ESC cultures remain highly undifferentiated, when serially passaged in suspension bioreactors for extended periods. Flow cytometry analysis and gene expression profiles of several pluripotency markers, in addition to colony and embryoid body (EB) formation tests were conducted at the start and end of the experiment and all showed that the ESC cultures remained highly undifferentiated over extended culture time in suspension. In vivo teratoma formation and in vitro differentiation into neural, cardiomyocyte, osteoblast and chondrocyte lineages, performed at the end of the long term culture, further supported the presence of functional and undifferentiated ESCs in the expanded population. Overall, this system enables the controlled expansion of highly pluripotent murine ESC populations.
Since the derivation of human embryonic stem (hES) cells, their translation to clinical therapies... more Since the derivation of human embryonic stem (hES) cells, their translation to clinical therapies has been met with several challenges, including the need for large-scale expansion and controlled differentiation processes. Suspension bioreactors are an effective alternative to static culture flasks as they enable the generation of clinically relevant cell numbers with greater efficacy in a controlled culture system. We, along with other groups, have developed bioreactor protocols for the expansion of pluripotent murine ES cells. Here we present a novel bioreactor protocol that yields a 25-fold expansion of hES cells over 6 days. Using immunofluorescence, flow cytometry, and teratoma formation assays, we demonstrated that these bioreactor cultures retained high levels of pluripotency and a normal karyotype. Importantly, the use of bioreactors enables the expansion of hES cells in the absence of feeder layers or matrices, which will facilitate the adaptation of good manufacturing process (GMP) standards to the development of hES cell therapies.
Mutations in the pathways regulating mammary epithelial stem cell (MESC) self-renewal and differe... more Mutations in the pathways regulating mammary epithelial stem cell (MESC) self-renewal and differentiation are currently hypothesized to result in uncontrolled cell division and, in turn, breast tumor formation. Although research is aggressively being pursued to understand how such pathways result in breast cancer formation, current studies have been greatly limited by MESC scarcity. To address this issue, this study has successfully developed large-scale expansion protocols for MESC through the subculture of murine mammary epithelial tissue aggregates, called mammospheres, in suspension bioreactors. Growth kinetics of mammospheres cultured in 125 mL suspension bioreactors and T-flasks were found to be comparable, achieving cell densities of 3.10 × 105 and 2.75 × 105 cells/mL, respectively. This corresponded to a 4-fold expansion over 8 days. Yields were also found to be strongly affected by liquid shear forces, where high agitation rates reduced overall cell numbers. Bioreactor cultures were scaled up to 1000 mL operating volumes, resulting in the production of 4.21 × 108 total cells (5.6-fold expansion) from a single passage. Furthermore, intermittent replacement of culture medium with fresh medium dramatically improved maximum cell densities, resulting in an 11-fold expansion, thereby enabling the generation of stem cells in quantities sufficient for standard biochemical and genetic analyses. After being cultured in suspension bioreactors for several passages, analysis by flow cytometry of Ki-67 revealed that 85% of the population was composed of proliferating cells. The successful development of expansion protocols for MESC aggregates in suspension bioreactors makes available experimental avenues that were not previously accessible for breast cancer research, thereby facilitating future investigations into elucidating the role of MESCs in breast cancer tumorigenesis.
Large-scale expansion of neural stem and progenitor cells will be essential for clinically treati... more Large-scale expansion of neural stem and progenitor cells will be essential for clinically treating the large number of patients suffering from neurodegenerative disorders such as Parkinson's disease. Other applications of neural stem cell technology include further research in areas such as neural development or drug testing. Neural stem cells can be grown in vitro as tissue aggregates known as neurospheres, and in the current study, experiments were performed to determine the spatial arrangement and behavior of the cells within the neurosphere structure. A protocol utilizing sulfonated lipophilic fluorescent dyes was developed to effectively label populations of neural stem and progenitor cells without compromising cell density during culture. Cells retained the labels for at least 7 days. Using the labeling protocol, we discovered that the cells within the neurospheres were mobile and, moreover, the cells on the periphery of the neurospheres could migrate into the center of the neurospheres. Most important, the mixing time of two merging neurospheres was observed to be the same order of magnitude as the neural stem cell doubling time (∼20 h). This study is the first to show that the neurosphere system is dynamic, and these results will serve as a stepping stone to more in-depth studies of the neurosphere microenvironment.
Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for r... more Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for regenerating tissues lost to injuries and degenerative diseases. For clinical implementation of this technology, a quality controlled, reproducible culture system is necessary for the expansion and differentiation of the cells. Used in many bioprocess applications, suspension bioreactors have gained considerable attention for the regulated large-scale expansion of cells. The current study presents a bioreactor process for the large-scale expansion of undifferentiated murine ESCs as aggregates. In this system, the level of ESC aggregation and differentiation was effectively controlled by adjusting shear forces and inoculation density, achieving a 31-fold expansion in 5 days. Pluripotency markers Oct-4, Nanog, SSEA-1, ALP, and rex-1 were assessed using flow cytometry analysis and gene expression profiles and showed that the undifferentiated nature of the cells within the ESC aggregates was maintained. Colony-forming efficiencies and embryoid body formation tests of the expanded cultures demonstrated that characteristic functional attributes of undifferentiated cells were not lost. Overcoming a major impediment in the area of ESC expansion, this study describes a successful process for the controlled and reproducible largescale expansion of ESCs using suspension culture bioreactors.
The aim of this study was to develop a scaleable process to expand pancreatic endocrine tissue (i... more The aim of this study was to develop a scaleable process to expand pancreatic endocrine tissue (i.e., aggregates or islet-like structures) in suspension bioreactors. Key issues addressed included (i) serum-free media, (ii) cell inoculation density, (iii) medium pH, and (iv) aggregate dissociation. Suspension bioreactors were inoculated with pancreatic neonatal tissue and operated under controlled conditions for a 9-day period. Medium studies showed that a new serum-free medium developed in our laboratory was capable of supporting endocrine cell expansion. An inoculation density of 127,000 cells/mL resulted in more than a 7.5-fold increase in the number of insulin-positive cells after 9 days. The resulting population consisted of single cells and many islet-like aggregates that contained all of the endocrine cell types (including insulin-positive, glucagon-positive, somatostatin-positive, and pancreatic polypeptide-positive cells). Furthermore, the cell aggregates exhibited a glucose-responsive behavior. This study represents a significant milestone on the path to the effective expansion of human islet-like tissue in bioreactors that may be used for cell therapy to treat Type 1 diabetes.
Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (N... more Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (NSC) have been determined. We examined suspension culture bioreactors of murine NSC, and concluded that the oxygen level should be kept high (20%), and the osmolarity of the medium should be kept low (below 400 mOsm/kg). The pH of the medium was found to have a large effect on cell proliferation, and the best growth characteristics were obtained within an optimum pH range of 7. 1 to 7.5. The inoculation conditions were also seen to have a large effect not only on the growth characteristics, but also on the number of cells that die in the initial stages of the culture. For large expansion of cells, low inoculum levels (10(4) cells/mL) and single-cell suspensions proved superior, whereas, for fast expansion of cells, higher inoculum levels (10(5) cells/mL) and spheroid inoculum forms were preferred. The inoculum temperature of the medium did not have a large effect on growth characteristics, but the pH greatly influenced cell proliferation. Inoculum pH levels should also be kept between 7.1 and 7.5. If these protocols are followed, high multiplication ratios and viabilities can be obtained in a 5-day batch suspension culture bioreactor run. A large number of cells could then be used in animal models for testing of neural drugs and in research and development toward cures for neurodegenerative disorders such as multiple sclerosis (MS) and Huntington&#39;s and Parkinson&#39;s disease. The results presented here also point the way toward studies on in vitro expansion of human neural stem cells.
The volume of heavy oil and bitumen in Alberta, Canada is estimated to be about 1.7 trillion barr... more The volume of heavy oil and bitumen in Alberta, Canada is estimated to be about 1.7 trillion barrels. The majority of the produced heavy oil and bitumen in Alberta is converted in surface upgraders to synthetic crude oil, a crude oil with API gravity typically between 31 and 33° API, which in turn can be converted to fuel, lubricant, and petrochemical products in standard refineries. To upgrade bitumen requires hydrogen. In current practice, much of this hydrogen is generated from catalytic steam reforming of methane together with the water–gas shift reaction. This means that heavy oil and bitumen upgrading, as is currently done, requires large amounts of natural gas to generate hydrogen. The potential for in situ generation of hydrogen by gasification of bitumen reservoirs offers an attractive alternative which can also have both economic and environmental benefits. For example, hydrogen generated from bitumen gasification can also be used for in situ upgrading as well as feedstock for ammonia and other chemicals. The water–gas shift reaction also generates carbon dioxide which could be potentially sequestered in an in situ gasification process so that emissions to the atmosphere are reduced. This technology provides a potential clean method to produce fuel and feedstock material from bitumen, a relatively “dirty” fuel and feedstock oil, in addition to more energy efficient ways of extracting in situ heavy oils. However, to design in situ bitumen gasification processes requires a reaction model that provides a reasonable representation of the gasification reactions. Here, a new kinetic model is developed to examine the potential for hydrogen generation from Athabasca bitumen. The kinetic model consists of thermal cracking, oxidation/combustion, hydrogen generation and hydrogen consumption reactions. A comparison of the simulation results and experimental data from the published literature reveal that the new model can predict hydrogen generation from gasification of methane, Athabasca bitumen, and coke.
Abstract With increased demand for fossil fuels of more than 50% in the next 25 years, several me... more Abstract With increased demand for fossil fuels of more than 50% in the next 25 years, several methods of either enhancing oil and gas production from existing fields or finding new fields and tackling unconventional sources, such as shale gas reservoirs, are currently underway. Microbially-generated methane gas is a significant portion of commercial gas production around the world. At least 20% of the world&amp;#x27;s methane originates from methanogens that reside within organic matter rich shales and coals. The metabolic ...
Extraction and upgrading of bitumen in Alberta, Canada uses large amounts of energy, generates hu... more Extraction and upgrading of bitumen in Alberta, Canada uses large amounts of energy, generates huge volumes of acid gas, consumes massive volumes of water, and is costly. Most bitumen produced in Alberta is converted in surface upgraders to synthetic crude oil (SCO), a 31 to 33°API oil product. Next, SCO is converted to transportation fuels and lubricants in conventional refineries. Bitumen upgrading requires hydrogen. Today, most of the hydrogen is produced by steam reforming of methane which requires huge amounts of methane. Alternatively, hydrogen can be generated by in situ gasification of bitumen. Gasification is potentially more energy efficient with reduced emissions since acid gases are sequestered to some extent in the reservoir. Also water usage is lowered and heavy metals and sulfur compounds in the bitumen tend to remain downhole. The overall objective of this research is to understand and optimize hydrogen generation from bitumen reservoirs. In situ technologies that convert bitumen to hydrogen will have direct application in bitumen upgrading, use as feedstock for ammonia and other chemicals, and may be a key step to start a hydrogen economy. One key step towards the design of in situ hydrogen generations processes is the construction of the reaction scheme together with the associated kinetic parameters. Here, a unified kinetic model that takes pyrolysis (thermolysis, thermal cracking), aquathermolysis, gasification, and combustion (oxidation) of Athabasca bitumen has been assembled. The model has been calibrated against 7 experimental and plant data sets (with 149 data points in total) and implemented in a thermal reservoir simulator. The unified kinetic model was developed by performing a global match of the reaction scheme and kinetic parameters against all available pyrolysis, aquathermolysis, gasification, combustion, and oxidation data.
The Canadian Journal of Chemical Engineering, 2002
Abstract A coiled quartz tubular reactor has been designed to measure the intrinsic reaction kine... more Abstract A coiled quartz tubular reactor has been designed to measure the intrinsic reaction kinetics for homogeneous reactions at high temperatures up to 1100 C. Actual gas residence times were less than 100 ms. A simple and well-studied test reaction (ie, the decomposition of nitrous oxide, N 2 O), with published intrinsic kinetics, was used to verify the operation of the experimental reactor. For this system, Peclet numbers (Pe= uL/DL) computed from experimental conversion data were greater than 1000, indicating that the ...
Journal of Petroleum Science and Engineering, 2012
ABSTRACT In Steam-Assisted Gravity Drainage (SAGD), high pressure, high temperature steam is inje... more ABSTRACT In Steam-Assisted Gravity Drainage (SAGD), high pressure, high temperature steam is injected into a bitumen-bearing oil sands formation. For most operations, the steam temperature ranges from about 200 to 260 °C and thus under these conditions, the bitumen undergoes hydrous pyrolysis, i.e. aquathermolysis, yielding acid gases such as hydrogen sulfide and carbon dioxide. Current SAGD thermal reservoir simulation models often take into account complex spatial heterogeneity of the geology and oil composition and the physics of heat transfer, multiphase flow, gas solubility effects, and viscosity variations with temperature, however, none so far in the literature take the detailed chemistry and reaction engineering of bitumen aquathermolysis during field scale SAGD into account. Here, we have added aquathermolysis reactions to a thermal reservoir simulation model to understand the reactive zones in the SAGD process and how the process generates acid gases via aquathermolysis. The results reveal that chemical reactions have a significant effect on the growth of the SAGD steam chamber and that the majority of the reactive zone is in the liquid pool that sits above the production well. The product gases of aquathermolysis reduce the efficiency of steam heat delivery capacity to the chamber edge. The results demonstrate that this effect can be reduced by lowering the steam injection pressure. This new model is capable of predicting acid gases like hydrogen sulfide, carbon oxides and fuel gases like hydrogen, methane as observed during SAGD field operations.
Summary Oil-sands reservoirs in western Canada hold more than 170 billion bbl of recoverable heav... more Summary Oil-sands reservoirs in western Canada hold more than 170 billion bbl of recoverable heavy oil and bitumen representing a significant source of unconventional oil. At in-situ conditions, the majority of this oil has essentially no initial mobility because of its high viscosity, which is typically in the hundreds of thousands to millions of centipoises. In steam-assisted gravity drainage (SAGD), steam injected into the formation heats oil at the edge of a depletion chamber, thus raising the mobility, ko/μo, of bitumen. Three main effects account for the increase of oil mobility. First, bitumen at steam temperature has viscosity typically less than 20 cp. Second, it is believed that shear, which is caused by thermal-expansion gradients, dilates the oil sand and causes enhanced permeability. Third, dilation at the chamber edge leads to smaller residual oil saturation (ROS). Because the production rate of SAGD is directly tied to the drainage rate of mobilized oil at the chamber...
Canadian Unconventional Resources and International Petroleum Conference, 2010
Understanding both physical and biological processes of gas generation and movement in immature o... more Understanding both physical and biological processes of gas generation and movement in immature organic matter rich shales is essential to optimize gas production from this resource. As yet, there is no complete description that accounts for the many gas transport modes in these systems. Field production data reveals that gas production from these reservoirs declines initially and then stabilizes after a specified time. The stabilized rate is controlled by the contributions of biogenic gas generation, desorption of gas from kerogen, diffusion and transport of gas through nanometer to potentially even micron scale pore systems in coarser grained intervals where physics of transport differ in mechanism. One factor that is ignored is the biogenic gas generation rate and whether this is significant in recharging the resource during a resource well lifetime. This paper presents a modified gas material balance to account for biogenic gas generation. The results of the theory are then comp...
Canadian Unconventional Resources Conference, 2011
In SAGD, steam is injected into a bitumen bearing oil sands formation. Steam temperature ranges f... more In SAGD, steam is injected into a bitumen bearing oil sands formation. Steam temperature ranges from about 200 to 260°C and at these temperatures, bitumen undergoes aquathermolysis yielding acid gases such as hydrogen sulphide and carbon dioxide. SAGD simulation models in the literature often account for spatial heterogeneity of the geology and oil composition and heat transfer, multiphase flow, gas solubility effects, and viscosity variations with temperature, however, none account for the chemistry of SAGD. Here, we consider aquathermolysis reactions to understand the reactive zones in the SAGD process and how the process generates acid gases via aquathermolysis. The results show that SAGD is both a physical and chemical-reactive process.
The volume of heavy oil and bitumen in Alberta, Canada is estimated to be about 1.7 trillion barr... more The volume of heavy oil and bitumen in Alberta, Canada is estimated to be about 1.7 trillion barrels. The majority of the produced heavy oil and bitumen in Alberta is converted in surface upgraders to synthetic crude oil, a crude oil with API gravity typically between 31 and 33°API, which in turn can be converted to fuel, lubricant, and petrochemical products in standard refineries. To upgrade bitumen requires hydrogen. In current practice, much of this hydrogen is generated from steam reforming of methane together with the water-gas shift reaction. This means that heavy oil and bitumen upgrading, as is currently done, requires huge amounts of natural gas to generate hydrogen. The potential for in situ generation of hydrogen by gasification of bitumen reservoirs offers an attractive alternative which can also have both economic and environmental benefits. For example, hydrogen generated from bitumen gasification can also be used for in situ upgrading as well as feedstock for ammonia and other chemicals. The water-gas shift reaction also generates carbon dioxide which could be potentially sequestered in an in situ gasification process so that emissions to the atmosphere are reduced. This study evaluates the effect of operating parameters such as temperature and pressure on the amount of hydrogen generated during in-situ combustion. Combustion of Athabasca bitumen incorporating thermal cracking, oxidation, and hydrogen generation and consumption reactions has been simulated. The results indicate ranges of temperature and pressure where bitumen combustion yields optimal hydrogen production. This research emphasizes the conditions at which hydrogen production can be maximized from the in-situ combustion of Athabasca bitumen. This technology provides a potentially clean method to produce fuel and feedstock material from bitumen, a relatively "dirty" fuel and feedstock oil, in addition to more energy efficient ways of extracting in-situ heavy oils.
International Petroleum Technology Conference, 2013
A continued increase in energy demand has amplified the significance of commercial heavy oil and ... more A continued increase in energy demand has amplified the significance of commercial heavy oil and bitumen recovery from complex carbonates formations such as the Grosmont Formation (OOIP ~ 406.5 billion barrels) in Alberta, Canada. To facilitate commercial development of bitumen carbonates, we have designed reservoir simulation models of complex carbonate reservoirs based on the concept of multiple interacting objects. Spatial distribution of different objects including fractures, vugs, breccia, and matrix are constructed by using stochastic methods with intensity functions derived from cores, logs, drilling and geologic data. Thermal reservoir simulations are conducted directly on realizations of these 'objects network' reservoir models. Although data from the highly fractured, karstified and vuggy bitumen-rich Grosmont Formation is used in this paper, this methodology is generic and applicable to other complex carbonate reservoirs. Results suggest that continuous type steam-based enhanced oil recovery (EOR) such as steam-assisted gravity drainage (SAGD) may not be best suited for bitumen recovery from complex carbonates.
Heavy-oil and Oil-sand Petroleum Systems in Alberta and Beyond, 2013
Currently, large-scale commercial in-situ recovery of bitumen from oil sands reservoirs is done b... more Currently, large-scale commercial in-situ recovery of bitumen from oil sands reservoirs is done by thermal gravity drainage oil sands recovery processes such as cyclic steam stimulation (CSS) and steam-assisted gravity drainage (SAGD). In these processes, steam is injected into the formation, which then heats bitumen until it is sufficiently mobile enough to be moved to the production well. The key goal of these processes is controlled and targeted steam delivery, really heat delivery, to the reservoir, and thus, both heat transfer and consequent bitumen mobilization are key controls on the performance of the processes. Here, we describe conductive and convective heat transfer at the edge of steam chambers and oil mobilization just beyond the edge of the chamber. The results demonstrate the complex interplay between heat transfer and oil mobilization: heat transfer controls the temperature profile beyond the edge of the steam chamber, which sets the oil viscosity profile, which in turn controls bitumen mobilization. Relative permeability, geomechanics, and geologic heterogeneity make the phenomena more complex. The discussion suggests that self-corrective robust recovery processes or dynamic well interventions such as smart wells that yield uniform steam chambers are required to ensure efficient heat transfer and oil mobilization.
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