Effects of Hydrodynamics on Cultures of Mammalian Neural Stem Cell Aggregates in Suspension Bioreactors

Abstract

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 × 10 6 cells/mL were achieved in a batch culture with viabilities remaining above 80% for the majority of the runs.

Large-Scale Expansion of Mammary Epithelial Stem Cell Aggregates in Suspension Bioreactors

“…Mammospheres were observed in suspension cultures 1 day after inoculation and continued to grow in size until they reached 40-80 µm in diameter, as seen in Figure 2. Similar observations have been found in a number of other aggregate suspension cultures, including neural stem cells, embryonic stem cells, and BHK cells, where aggregates rapidly formed and continued to grow in size throughout the growth phase (21,32). Though mammospheres were also observed in stationary cultures, they were found to be much smaller and resembled cell clusters rather than spherical aggregates.…”

“…This effectively results in reducing aggregate size with the removal of cells along its surface. This phenomenon has proven useful in neurosphere cultures, as a means of avoiding potential mass transfer issues near aggregate centers (21). However, shear sensitivity of cells must also be accounted for, since such conditions could potentially result in shear stresses that compromise cell viability (31).…”

“…Pertinenent vessel dimensions are also provided in Table 1. Published values of medium density and viscosity were used for maximum shear stress calculations (21).…”

Expansion of Human Neural Precursor Cells in Large‐Scale Bioreactors for the Treatment of Neurodegenerative Disorders

“…Thus, it is of great importance to control the size of aggregates to avoid the development of necrotic cores caused by inadequate oxygen levels. 28 Previous studies involving the expansion of murine neural precursor cells (mNPCs) have shown that maintaining the maximum shear stress (s max ) of the impeller tip between 0.3 and 0.75 Pa can control the size of aggregates while minimizing damage to the cells. 28,29 Mass transfer rates and culture hydrodynamics in suspension bioreactors are dramatically influenced by vessel geometry, impeller design, and the presence of measuring probes.…”

“…32,37 Previous studies on mouse NPCs have shown that shear can be used to control the size of the aggregates formed in suspension culture. 28 To investigate the effect of liquid shear on hNPC aggregate production, hNPCs at passage level 10 were inoculated into standard 125-mL suspension bioreactors operated at 70, 100, or 130 rpm. Using the Kolmogoroff theory of turbulent eddies 37 and the Nagata correlation, 38 the equivalent maximum shear stress for agitation rates of 70, 100, and 130 rpm was calculated to be 0.35, 0.58, and 0.82 Pa, respectively.…”