Effects of Hydrodynamics on Cultures of Mammalian Neural Stem Cell

Oct 13, 2001 - Although difficult to grow in bioreactors, neural stem cells can be expanded in carefully designed media as aggregates of brain tissue...
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Effects of Hydrodynamics on Cultures of Mammalian Neural Stem Cell Aggregates in Suspension Bioreactors Arindom Sen, Michael S. Kallos, and Leo A. Behie* Pharmaceutical Production Research Facility (PPRF), Faculty of Engineering, University of Calgary, Calgary, Alberta, Canada T2N 1N4

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 shear and oxygen transfer rate, in bioreactors. This is very important because large aggregates can develop necrotic centers of dead cells due to transport limitations of key nutrients. Manipulation of the agitation rate allowed us to control the average aggregate diameter to 150 µm, below levels where necrosis would occur. Moreover, for the best conditions, viable stem cell densities of 1.2 × 106 cells/mL were achieved in a batch culture with viabilities remaining above 80% for the majority of the runs. 1. Introduction

Table 1. Current Prevalence and Health Care Costs of Neurodegenerative Disorders in North America27

The growing need for new disease treatments in a rapidly aging population has fueled the examination of cellular therapies that could remedy the cause of diseases rather than pharmacological treatments, which generally treat the symptoms. This is particularly true for neurodegenerative diseases of the central nervous system. Table 1 shows the estimated number of individuals in North America afflicted by neurodegenerative diseases, and the staggering economic cost associated with treating them. Neural stem cells (NSC) are gaining importance as potential therapeutic agents to treat diseases such as Parkinson’s and multiple sclerosis.1,2 These undifferentiated cells, with an extended proliferative capacity, have the ability to form all of the major cell types in the central nervous system (CNS), including neurons, astrocytes, and oligodendrocytes. In mouse models of Parkinson’s disease, dopaminergic neurons derived from ex vivo expanded human NSC have been transplanted into the affected region of the brain. These cells were shown to be incorporated into the striatal architecture as well as induce functional recovery.3 Other researchers have established nontransformed human NSC lines, which when transplanted into the brain of adult rodents, subsequently migrated from the site of injection and differentiated into neurons and astrocytes.4 These studies demonstrate the efficacy of NSC as a powerful treatment avenue for neurodegenerative disorders. Gene therapy using NSC is also developing into a path of treatment for CNS disorders.5 The use of these primitive stem cells is presently restricted to experimental studies, but promising results have been obtained in murine models of Parkinson’s disease3,6 and Huntington’s disease.7 It is anticipated that large numbers of cells will be required for research, drug testing, and transplantation. For biomedical engineers * To whom correspondence should be addressed. E-mail: [email protected]. Fax: (403) 284-4852.

disease/disorder

health care costs estimated cases (U.S.$ billions/year)

Alzheimer’s disease stroke brain and spinal injuries Parkinson’s disease multiple sclerosis other

4 400 000 2 200 000 1 125 000 550 000 300 000 21 843 000

74.0 33.0 58.3 20.0 10.7 26.2

total

30 418 000

222.2

in this burgeoning field, the challenge now is to develop bioreactor protocols to grow and expand these cells on a large scale. NSC. Stem cells are tissue-specific pluripotent cells. They divide to give rise to progenitor cells, which then further divide to generate fully functional, terminally differentiated cells. As a group, stem cells and progenitor cells are referred to as precursor cells. Stem cells reside in tissues such as bone marrow (hematopoietic stem cells) and skin (epithelial stem cells), which require constant regeneration as a result of damage or daily wear and tear. Stem cells replace the parenchymal cells of each tissue, as necessary, for the lifetime of the organism. Until recently, stem cells were thought not to exist in the CNS because neural tissue does not fully regenerate or repair itself after injury. However, in 1992, Weiss and Reynolds isolated growth-factorresponsive neural precursor cells from both murine adult and embryonic sources, which were later proven to have all of the characteristics of stem cells.8,9 Since then, the field of NSC biology has exploded.2 Human NSC have been induced to expand ex vivo by both epigenetic (i.e., cell division stimulated by the addition of growth factors) and genetic means.4,10-12 In these studies, the NSC grew as aggregates and were passaged for up to a year in stationary culture. The supply of human cells is very limited, and there are a number of ethical concerns that need to be addressed when using this source of tissue. Because murine cells are genetically similar to human cells13 and are readily

10.1021/ie001107y CCC: $20.00 © 2001 American Chemical Society Published on Web 10/13/2001

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Figure 1. Potential applications of neural stem cell (NSC) technology in the treatment of neurodegenerative disorders, brain injuries, and spinal cord injuries. Sources of neural stem cells include the adult central nervous system (CNS), fetal central nervous system, and embryonic stem cells (ESC).

available, it is advantageous to first develop expansion protocols with murine cells and then apply the new protocols to human cells. The present experiments have been performed using a nontransformed murine NSC line expanded in bioreactors using epigenetic means. Therapeutic Applications. Figure 1 displays a hypothetical NSC treatment pathway. NSC could be procured from the adult CNS, fetal CNS or derived from embryonic stem cells (ESC). Because of moral and ethical concerns, fetal and embryonic sources would be used sparingly to establish cell banks. Regardless of the source, NSC would then need to be expanded ex vivo to give rise to populations of stem cells, progenitor cells, and mature cells, depending on the application. These cells could be used for drug testing or in vivo therapies such as gene therapy, drug delivery through engineered cells, or transplantation. Autologous NSC transplantation represents the most appealing therapy, as the patient’s own cells would be used to repair damaged or diseased portions of their CNS. In this case, effective expansion of very low initial numbers of cells would be required. Bioreactor Scale-Up: Suspension Culture. The growth of NSC in stationary culture (tissue culture flasks) does not lend itself to large-scale applications. This type of culture is labor-intensive, expensive and does not allow for the control of important physiochemical parameters such as dissolved oxygen and pH. A suspension culture in computer-controlled bioreactors offers the advantages of homogeneity and strictly controlled conditions, as well as being compact and scalable. A continuous culture system that is attractive for the growth of NSC is a perfusion culture, wherein fresh

medium is continuously fed into the bioreactor and the spent medium is continuously removed. A perfusion culture is necessary in order to maintain constant conditions in the bioreactor. In a continuous perfusion culture, nutrient and growth factor limitations do not occur, and toxic metabolic byproducts are constantly removed. The growth of NSC in aggregates is advantageous, because the aggregates can be separated from the exiting medium by simple sedimentation, and the complex, and often very expensive, problem of retaining the cells in the bioreactor is solved.14 Cell Growth in Aggregates. NSC cultures are typically inoculated as a single-cell suspension, and population expansion occurs through subsequent cell division. As the cells divide, the daughter cells remain attached to each other because of the production of “sticky” extracellular matrix (ECM) molecules. This eventually results in the formation of spherical aggregates made up of hundreds or thousands of cells. Cells within aggregates receive essential substrates, such as oxygen, nutrients, and growth factors, from the bulk medium and eliminate wastes into the bulk medium through the process of diffusion.15 Substrates diffuse into the aggregate from the bulk medium through layers of cells and are consumed as they pass the cells in each layer. As a result, concentration gradients exist from the outer edge of the aggregate to the center, and these will result in different microenvironments for cells throughout the aggregate. Cells at the outer edge of the aggregate will be exposed to higher nutrient, oxygen, and growth factor concentrations than those cells a few layers under the surface of the aggregate. This leads to varying growth rates and nutrient consumption rates within the aggregate16 and may result in stem cell differentiation. It is also possible that cells at the center of very large aggregates (diameter greater than 300 µm) will experience extreme limitations in nutrients and oxygen, leading to cell death, a condition known as necrosis.17 To avoid the formation of necrotic centers, it is desirable to control the size of the aggregates. The transportation of substrates such as oxygen and nutrients to cells within aggregates is similar to the classic chemical engineering problem of diffusion and reaction in spherical catalyst pellets. The reaction in this case is the rapid consumption of the substrate by the cells, and so the process is mass-transfer-limited. The application of Fick’s law of diffusion shows that the maximum diffusion distance of oxygen through tissue [cell densities of (0.5-1.0) × 109 cells/mL] is between 125 and 150 µm.18 This means that cells beyond this distance will not have an adequate supply of oxygen. Moreover, the maximum distance of any cell to a capillary in mammals is less than 150 µm, and therefore this is arguably the maximum safe distance any cell should be located from the fresh medium.19 Aggregates (spheroids) have been studied extensively as 3D models of in vivo tumor growth.15 They are used to test the efficacy of novel cancer drugs, as well as schedules and dosages of radiation therapy, and to predict the profiles of oxygen, pH, nutrients, and waste products within tumors. Nutrient kinetics, spheroid growth rates, and viability have all been correlated with aggregate size. Aggregate Size Control. Control of aggregate diameter has been examined for many industrially important mammalian cell lines including BHK cells,

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CHO cells, and 293 cells.20-22 These cell lines are used for the production of valuable biological molecules such as vaccines. Shear, media additives, and growth conditions have all been used effectively to maintain aggregate diameters below levels where necrosis would be expected to occur.22 We have previously shown that shear in suspension bioreactors can be used to control the aggregate diameter of mammalian NSC23 and that murine NSC can be passaged for extended periods of time in the suspension culture with no loss of proliferative potential or stem cell characteristics.24 However, the previous work23,25 was performed with a proprietary medium (which is no longer available) as part of a contract with a North American biotechnology company. The present work was performed using a new, improved NSC medium (PPRF-m4), which is based in part on past work in our PPRF laboratory.26 In this study, we show that we can control the aggregate diameter while maintaining high cell viabilities. 2. Materials and Methods Cell Lines. The cell line used in experiments was a murine NSC line obtained as a gift from the laboratory of Professor Sam Weiss at the University of Calgary (Canada). Primary cultures of murine cells were derived as outlined by Reynolds and Weiss.8 Cells were obtained from the thalamus region of the forebrain of Embryonic Day 14 (E14) CD1 albino mouse embryos. Cells were obtained as a primary culture and passaged immediately into PPRF-m4 medium (passage 1). Experiments were initiated with passage level 2 cultures. Medium Preparation. The medium used to conduct all experiments was PPRF-m4, a new medium developed in our laboratory. The medium used for the growth of murine cells was vacuum filtered through a 0.22 µm bottle-top filter (Falcon 7105) prior to use. The basic culture medium was composed of the basal media DMEM (catalog no.12100-046) and F12 (catalog no. 21700-075) from Gibco BRL in a 1:1 mixture. Supplements to the basal media mixture included 5 mM HEPES (H-9136), 0.4% glucose (G-7021), 1.73 g/L sodium bicarbonate (S-5761), 2 mM glutamine (25030016), 25 mg/L insulin (I-5500), 20 nM progesterone (P6149), 9 mg/L putrescine (P-7505), 0.05 g/L transferrin (T-2252), 30 nM sodium selenite (S-9133), and 20 µg/L EGF (100-15). All concentrations represent final concentrations in the medium. A proprietary supplement mixture developed in our laboratory was also added to the medium. All of the supplements were obtained from Sigma with the exception of glutamine (Gibco BRL) and EGF (Peprotech Inc.). Cell Handling Procedures. All cell-handling procedures, unless otherwise noted, were performed in a sterile laminar flow hood to prevent contamination by bacteria, fungus, or mycoplasma.25,27 Cell Passaging: Standard Procedure for a Stationary Culture. Stocks of murine cells were maintained in 75 cm2 tissue culture flasks (T-flasks) and passaged every 4 days when the aggregates reached diameters near 200 µm. This ensured that large aggregates would not form and all cells in the center would have access to sufficient nutrients. Cell suspensions were harvested from T flasks by gently tapping the sides of the T flask to dislodge any attached spheres and then pipeting the contents into a 15 mL centrifuge tube. The cell suspension was centrifuged for 10 min at 1000 rpm

(173g), and most of the supernatant was removed. Approximately 200 µL (including the cell pellet) was left in the 15 mL centrifuge tube. A homogeneous singlecell suspension was obtained for inoculum by mechanically dissociating (triturating) the pellet that remained with a 200 µL pipet (VWR Brand tips, 53509-000). The pipet was set at 30 µL less than the volume of the pellet, and the cells were triturated 30 times. While triturating, the tip was held pressed against the bottom of the centrifuge tube at an angle, and the sides of the centrifuge tube were periodically rinsed with the cell suspension to dislodge any attached spheres. Care was taken not to form bubbles in the cell suspension. After the spheres had been dissociated, a small sample was taken for counting, and then the cells were inoculated into 15 mL of preincubated PPRF-m4 medium at 0.75 × 105 cells/mL in 75 cm2 T-flasks. The T-flasks were placed in a 37 °C incubator with a water-saturated atmosphere containing 5% CO2. The elevated CO2 concentration in conjunction with the buffers in the medium ensured that the cells would be maintained within a physiological pH range for the duration of the culture. Cells were passaged 4 days later. Cell Passaging: Standard Procedure for a Suspension Culture. In all bioreactor experiments, murine cells were maintained in 125 mL bioreactors (spinner flasks) and passaged every 4 days unless otherwise noted. The contents of a spinner flask were passaged by pipeting four 10 mL aliquots from the spinner flask and placing each aliquot in a separate 15 mL centrifuge tube. The aliquots were then centrifuged for 10 min at 1000 rpm (173 g), and most of the supernatant was subsequently removed. Approximately 200 µL (including cell pellet) was left in each 15 mL centrifuge tube. A homogeneous single-cell suspension was obtained in each tube by triturating the contents as per the mechanical dissociation instructions described in the stationary culture procedure. The contents of the four centrifuge tubes were then consolidated into a single 15 mL tube, and a small sample was taken for counting. The cells were inoculated at 0.75 × 105 cells/mL into 100 mL of preincubated PPRF-m4 medium in a 125 mL spinner flask. All spinner flasks were placed in a 37 °C incubator with a water-saturated atmosphere containing 5% CO2. Cell Culture Equipment. (a) Tissue Culture Flasks. Tissue culture flasks were obtained from Life Technologies (Gibco, Gaithersburg, MD). The Nunc 75 cm2 Easyflask (Nunc catalog no. 156499) was used for the maintenance of cell stocks. (b) Bioreactors/Spinner Flasks. All suspension culture experiments were performed in 125 mL bioreactors (spinner flasks) from Corning (Corning Glass, Corning, NY). During experiments, the flasks were placed on Thermolyne Cell-Gro slow-speed magnetic stirrers set at speeds of 60-100 rpm (Thermolyne, Dubuque, IA). The inner surface of the flask and the outer surface of the magnetic stir bar were siliconized with Sigmacote (Sigma, St. Louis, MO) prior to use to prevent the cells from sticking to the surface. Analytical Procedures. (a) Spinner Flask Sampling. All spinner flask experiments were performed in duplicate unless otherwise noted, and samples were taken daily. Samples were obtained from spinner flasks using the following procedure. First, the spinner flask was removed from the incubator and placed on a Thermolyne magnetic stirring plate just outside the

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laminar flow hood. Second, the spinner flask was brought into the hood, a 2 mL sample was aseptically removed using a 5 mL plastic pipet (Falcon, 7543), and the spinner flask was placed back on the spinner plate. Finally, the spinner flask was returned to the incubator. This procedure minimized the amount of time the spheres were allowed to settle. If left for an extended period of time, nonsuspended spheres could have adhered to one another, resulting in the formation of extremely large, unwanted aggregates. A small sample (100 µL) was taken from the 2 mL sample for visual observations prior to centrifugation. (b) Cell Counts and Viabilities. The cell density was determined using a hemocytometer. Viability was determined using the standard trypan blue exclusion test (Sigma Chemicals T8154). Cell counts were performed in duplicate. Spherical aggregates were dissociated mechanically and then a sample was taken for counting. The sample was diluted in Ca2+- and Mg2+free phosphate-buffered saline (0.2 g/L KCl, 0.2 g/L KH2PO4, 8.0 g/L NaCl, and 2.16 g/L Na2HPO4‚7H2O, all from Sigma) and then stained with 0.1% trypan blue. (c) Spheroid Size Distributions. The aggregate diameter was determined by taking photomicrographs of cell samples and measuring two perpendicular diameters on a minimum of 20 aggregates. The average diameter for each aggregate was calculated, and then the mean for the sample was determined. Aggregates of less than 35 µm in diameter were not considered in the calculations, because they were generally single cells or doublets that were too small to accurately measure. 3. Results and Discussion Small bioreactors such as spinner flasks utilize a magnetic impeller to suspend cells and eliminate nutrient and oxygen concentration gradients within the medium. This ensures that cells are exposed to wellmixed, uniform conditions throughout the bioreactor. However, the mixing action results in the development of liquid shear in the medium. One of the objectives of this study was to examine the effect that this shear has on the size of NSC aggregates in a suspension culture in our new PPRF-m4 medium. Spinner flasks were inoculated with single cells at a density of 0.75 × 105 cells/mL and then agitated at 60, 80, or 100 rpm in a 37 °C humidified incubator for several days. During the culture period, the spinner flasks were sampled daily, and the viable cell density, viability, and average aggregate diameter were measured. In addition, nutrient and waste-product levels in the medium were also monitored. Effect of Shear on Cell Expansion. Figure 2 shows the viable cell density and cell viability of NSC cultures at 60, 80, and 100 rpm over the course of a batch run. In each case, the cell density decreased after inoculation, indicating a lag phase. Following this, the cells exhibited exponential growth for 6 days at 60 rpm and 5 days at 80 and 100 rpm. Although the length of the exponential growth phase was longer, the growth rate of 0.0198 h-1 at 60 rpm was significantly lower than the growth rate of 0.0424 h-1 at 80 rpm and 0.0328 h-1 at 100 rpm. The maximum viable cell densities in the 80 and 100 rpm flasks were similar (1.2 × 106 cells/mL) and much higher than that of the 60 rpm flasks (0.73 × 106 cells/mL). The initial viability in each flask sharply increased between the time of inoculation (68%) and the time the first sample was taken (89%) approximately 1 h later

Figure 2. Effect of the agitation rate on the cell density and viability of NSC in batch suspension bioreactors. Cells were grown in 125 mL spinner flasks with 100 mL of a PPRF-m4 medium in a 37 °C incubator with a water-saturated atmosphere containing 5% CO2. Bioreactors were inoculated with single cells at passage level 2 at 0.75 × 105 cells/mL. All data points represent the average of duplicate spinner flasks each counted twice. Error bars represent the standard error for each measurement.

(see Figure 2). The low inoculation viability can be attributed to the mechanical dissociation procedure that was used to produce a single-cell suspension for the inoculum from a culture of aggregates. Passing the aggregates repeatedly through a 200 µL pipet tip has been shown to result in unavoidable cell damage and death.24 The jump in viability (68-89%) was due to the physical destruction of a large number of intact dead cells in the inoculum by the shear present in the newly inoculated spinner flasks. The destruction of the dead cells resulted in a suspension of debris and single healthy cells. After the initial sharp increase, the viability remained constant for 3-4 days and then declined steadily at all agitation rates, although it should be noted that the viability remained considerably higher in the 100 rpm flasks (see Figure 2). Overall, the aggregates observed in the 60 and 80 rpm spinner flasks were larger than those in the 100 rpm flasks (see Figures 3 and 4). It is possible that the larger aggregate size leads to some degree of necrosis within the aggregates, thereby resulting in lower viabilities. However, the mechanical dissociation procedure used during the counting protocol may have also been a contributor. The larger aggregates would have experienced greater levels of shear when being passed through a 200 µL tip repeatedly, resulting in artificially low viabilities. This effect would have been greatest in the 60 and 80 rpm flasks because they had considerably larger aggregates than the 100 rpm flasks (see Figures 3 and 4). Effect of Shear on the Aggregate Diameter. The average aggregate diameter for all agitation rates is shown in Figure 3. After the first day in the culture, the cells at the lowest agitation rate (60 rpm) had already formed aggregates with a mean diameter of 125 µm, while those at the highest agitation rate (100 rpm) were only an average of 50 µm in diameter. Because the overall cell density did not increase during the first day in the culture, it can be assumed that the aggregates were composed primarily of the inoculum cells sticking together. It should also be noted that the

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Figure 3. Effect of the agitation rate on the mean aggregate diameter of NSC in batch suspension bioreactors. Also shown is the standard deviation in the mean aggregate diameter. All data points represent the average of duplicate spinner flasks each counted twice. Error bars represent the standard error for each measurement.

Figure 4. Effect of shear (agitation rate) on the maximum aggregate diameter of NSC in batch suspension bioreactors. All data points represent the average of duplicate spinner flasks each counted twice. Error bars represent the standard error for each measurement.

number of aggregates in the 60 rpm flask was lower than those in the 80 and 100 rpm flasks. This observation may aid in explaining why the measured exponential growth rate at 60 rpm was lower than that at 80 or 100 rpm. It is possible that once aggregates form, the cells within the aggregate divide at a much lower rate or remain relatively quiescent because of contact inhibition, whereas the cells at the surface continue to rapidly divide. If that is the case, the total outer surface area of the aggregates at 60 rpm would be lower than that at 80 or 100 rpm, and thus fewer cells would be dividing initially in the 60 rpm culture. As the runs progressed, the shear in the 100 rpm flasks was sufficient to control the mean diameter below 150 µm, while the aggregates exposed to 60 rpm continued to grow, reaching a mean diameter of over 300 µm by the end of the run (see Figure 3). These results agree with previous work, where the mean

diameter of NSC in suspension bioreactors was controlled below 125 µm at 100 rpm in a proprietary medium.23 It is likely that the higher agitation rate benefited the cells in two ways. First, the creation of a more intense liquid shear field resulted in lower mean aggregate diameters and therefore minimized the magnitude of any diffusional gradients within the aggregates. Second, the higher agitation rate improved the mass-transfer characteristics within the bioreactor. The mass transfer of oxygen from the gas phase to the liquid phase (i.e., to the cells) occurs primarily through diffusion from the headspace gas into the medium. The masstransfer coefficient increased by 49% (i.e., kla ) 1.562.33 h-1) when the agitation rate was increased from 60 to 100 rpm. The decreased mass transfer of oxygen at the lower agitation rate may have contributed to the lower NSC viabilities observed at 60 rpm. In the present study, not only was the mean diameter significantly lower at 100 rpm, but also the range of observed aggregate sizes was much narrower, indicating tighter control of the aggregate diameter. This can be seen in Figure 3, where the standard deviation in the mean aggregate diameter is shown for the duration of the batch experiment. For the entire run, most of the aggregates were within 40 µm of the mean at 100 rpm, while at 60 rpm most of the aggregates were within approximately 300 µm of the mean at 60 rpm. While mammalian cells are being cultured in vitro, it is desirable to have almost all of the cells in the bioreactor exposed to the same conditions. In the case of NSC, if all of the aggregates have the same controlled diameter, then the majority of the cells will remain within a range where they do not experience nutrient, oxygen, or growth factor limitations. The conditions within these aggregate microenvironments could then be optimized to maximize cell expansion and minimize differentiation by manipulating the medium composition and physiochemical conditions in the bioreactor. This is desirable in designing a reproducible, quality-controlled procedure for expanding mammalian NSC. In the flasks operating at 60 rpm, the presence of a wide range of aggregate diameters would result in many different microenvironments. Within very large aggregates, a number of cells would not be as healthy and may even differentiate or die. This is undesirable because the objective is to expand healthy stem cells and not unhealthy, differentiated cells. The maximum aggregate diameter attained over the entire course of each of the cultures is shown in Figure 4. This is an important variable to measure because it represents the upper limit of aggregate growth. The aggregates at 100 rpm did not exceed 250 µm in diameter, while those at 80 rpm exceeded 400 µm and those at 60 rpm approached 1000 µm. At sizes above 300 µm, severe nutrient limitations could occur. Indeed, when viewed under the microscope, the larger aggregates had an altered appearance at their center when compared to smaller aggregates. It is not known whether this histological change is due to necrotic, apoptotic, or differentiated cells. Work is currently underway in our laboratory to answer this question. Effect of Shear on Aggregate Size Distributions. Aggregate size distributions for NSC under different shear conditions are shown in Figures 5-7. In Figure 5, at 20.5 h following inoculation, most of the cells at 80 and 100 rpm were in aggregates below 100 µm. For the cells in the 60 rpm cultures, over half of the sampled

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Figure 5. Frequency distribution histogram for aggregates from flasks at 60, 80, and 100 rpm at a sampling time of 20.5 h postinoculum. All data points represent the average of duplicate spinner flasks each counted twice.

Figure 6. Frequency distribution histogram for aggregates from flasks at 60, 80, and 100 rpm at a sampling time of 68 h postinoculum. All data points represent the average of duplicate spinner flasks each counted twice.

aggregates had diameters above 121 µm and approximately 25% were above 240 µm. By 68 h postinoculation, 100% of the aggregates in the 100 rpm culture were between 41 and 160 µm in diameter, as shown in Figure 6. The majority of aggregates in the 80 rpm culture were distributed between 121 and 160 rpm, although some had diameters below 40 µm and others had diameters above 300 µm. In the 60 rpm cultures, over 40% of the aggregates had diameters above 300 µm. It is interesting to note that the percentage of sampled aggregates in the 60 rpm culture that were below 40 µm in diameter almost doubled to 18% between 20.5 and 68 h and that this value was even higher at 116.5 h (see Figure 7). It is also noteworthy that at each of the postinoculation times shown for 60 rpm, although there were aggregates

Figure 7. Frequency distribution histogram for aggregates from flasks at 60, 80, and 100 rpm at a sampling time of 116.5 h postinoculum. All data points represent the average of duplicate spinner flasks each counted twice.

at each end of the measured diameter spectrum, there were very few, if any, aggregates between 161 and 240 µm. This is contrary to what was observed in the 80 rpm culture at 116.5 h where the majority of sampled aggregates had diameters between 161 and 260 µm. With the exception of the first 48 h following inoculation in the 80 rpm culture, there were hardly any aggregates observed below 80 µm, although single cells were visible. This was also the case for the 100 rpm culture. It is unknown why these single cells did not start forming aggregates immediately. It was previously shown that single cells shed from NSC aggregates could indeed go on to form new aggregates when inoculated into a fresh medium.24 It is also important to note that, in the 100 rpm culture at 116.5 h, approximately 75% of the sampled aggregates had diameters between 101 and 180 µm and that the maximum diameter size observed was 230 µm. Taken together, the data presented in Figures 5-7 suggest that at 60 rpm most of the single cells in the inoculum immediately form small to mid-sized aggregates because the level of shear is too low to break them apart once they have attached to one another and that these aggregates then agglomerate with one another to form large aggregates. Indeed, visual microscopic observations of the aggregates early on in these cultures revealed that most of the large aggregates were not spherical but rather the amalgamation of two or three smaller aggregates. The single cells shed from these very large aggregates are able to form more aggregates, as is evidenced by the increasing number of aggregates below 80 µm at 68 and 116.5 h. The single cells in the inoculum of the 80 and 100 rpm cultures also showed a tendency to aggregate following inoculation, although not nearly to the same size as that observed in the 60 rpm culture, because of the higher level of shear. The higher shear level at 80 and 100 rpm also prevented the amalgamation of small and mid-sized aggregates into large aggregates, thereby controlling the maximum aggregate diameter size early on in the

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Figure 8. Average cell packing density for aggregates from flasks at 60, 80, and 100 rpm. All data points represent the average of duplicate spinner flasks each counted twice. Error bars represent the standard error for each measurement.

culture (see Figure 4). However, the fact that 25% of the sampled aggregates in the 80 rpm culture had diameters over 300 µm at 116.5 h and over 90% of the aggregates were over 180 µm would suggest that an agitation rate as low as 80 rpm is not adequate at controlling the aggregate diameter within an acceptable range (see Figure 7). This is in contrast to the 100 rpm culture at 116.5 h, where the majority of sampled aggregates were between 100 and 180 µm in diameter, only 6% of the aggregates had diameters above 220 µm, and the maximum diameter observed was approximately 230 µm. Cell Packing Density (PD) in Aggregates. The cell PDs of aggregates in the 60, 80, and 100 rpm cultures are shown in Figure 8. PD is computed as the total number of viable and dead cells per unit aggregate volume.28 From Figure 8 it is evident that, as the agitation rate of the culture increased from 60 to 100 rpm, the PD increased in a linear manner from 0.51 × 109 to 1.00 × 109 cells/mL of aggregate. These values are similar to those found in tumor spheroids, which have PDs of 0.5 × 109 cells/mL of aggregate,29 and mammalian tissue, which has a PD of between 1 × 109 and 3 × 109 cells/mL of aggregate.18 It is unknown why the PD increased with the agitation rate. It is possible that the PD is affected by the mode of aggregate growth. Aggregates that form in large part because of consolidation of single cells may exhibit a lower PD than aggregates that form primarily because of cell division. As previously discussed, single cells in the 60 rpm culture initially consolidated to form much larger aggregates than those observed at 100 rpm, possibly contributing to the lower overall PD observed within the 60 rpm culture. Furthermore, under different shear conditions it is likely that the amount and type of ECM produced by the NSC will vary, thereby influencing the overall PD in the aggregate. It was also observed that, for the most part, the PD of NSC aggregates did not change considerably during the logarithmic phase of growth, and no correlation was found with the aggregate diameter (R2 ) 0.05; data not shown). Studies on tumor spheroids also found no correlation between the spheroid diameter and the PD.29

Medium Analysis. Media formulation has been shown to greatly influence the formation of aggregates in a suspension culture.22 The current study was performed with a new medium formulation (PPRF-m4). Glucose and lactate measurements confirmed that there were no nutrient limitations in the bulk medium for the 60, 80, and 100 rpm flasks. The cells consumed less than 50% of the glucose supplied (data not shown), and the bulk medium lactate levels did not exceed 1.84 g/L in the 60 rpm flasks, 1.83 g/L in the 80 rpm flasks, and 1.46 g/L in the 100 rpm flasks. The pH of the 60 rpm culture dropped from 7.3 to 7.1 during the course of the experiment, which has previously been shown to be within the acceptable range for mammalian NSC.30 It is possible that the higher levels of lactate present at lower agitation rates may have been due, in part, to reduced oxygen mass transfer. Generally, lactate levels below 2.0 g/L are not expected to be detrimental to the survival of mammalian cells.31 However, it is unknown if the lactate levels within the aggregates were higher than those in the bulk medium. 4. Conclusions The objective of this study was to examine the control of the NSC aggregate size by changing the liquid shear field in batch culture bioreactors containing PPRF-m4, a new medium developed in our laboratory. As a result of the higher viabilities, considerable expansion of cells, and tightly controlled aggregate diameter, 100 rpm was chosen as the optimum agitation rate in the PPRF-m4 medium. Accordingly, the bioreactor protocols developed allowed us to control the mean aggregate diameter to 150 µm, below levels where necrosis would occur. Moreover, for the best conditions, viable stem cell densities achieved were 1.2 × 106 cells/mL in a batch culture. This study represents an important step on the path to large-scale expansion of mammalian NSC. Acknowledgment This research was funded, in part, by grants from a Canada Research Chair (L.A.B.) and the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors thank Professor Sam Weiss for his generous donation of cells and acknowledge the technical support of Wendy J. Paramchuk (M.Sc.) and Andrea C. Behie (M.Sc.) at PPRF. Literature Cited (1) Svendsen, C. N.; Smith, A. G. New Prospects for Human Stem-cell Therapy in the Central Nervous System. Trends Neurosci. 1999, 22 (8), 357-364. (2) Weiss, S. Pathways for Neural Stem Cell Biology and Repair. Nat. Biotechnol. 2000, 17, 850-851. (3) Studer, L.; Tabar, V.; McKay, R. D. G. Transplantation of Expanded Mesencephalic Precursors Leads to Recovery in Parkinsonian Rats. Nat. Neurosci. 1998, 1 (4), 290-295. (4) Vescovi, A. L.; Gritti, A.; Galli, R.; Parati, E. A. Isolation and Intracerebral Grafting of Nontransformed Multipotential Embryonic Human CNS Stem Cells. J. Neurotrauma 1999, 16 (8), 689-695. (5) Snyder, E. Y.; Park, K. I.; Flax, J. D.; Liu, S.; Rosario, C. M.; Yandava, B. D.; Aurora, S. Potential of Neural “Stem-Like” Cells for Gene Therapy and Repair of the Degenerating Central Nervous System. Adv. Neurol. 1997, 72, 121-132. (6) Yandava, B. D.; Billinghurst, L. L.; Snyder, E. Y. “Global” Cell Replacement is Feasible via Neural Stem Cell Transplantation: Evidence From the Dysmyelinated shiverer Mouse Brain. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7029-7034.

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Received for review December 15, 2000 Revised manuscript received August 29, 2001 Accepted August 30, 2001 IE001107Y