Developmental Brain Research 134 (2002) 103–113 www.bres-interactive.com Interactive report Expansion of mammalian neural stem cells in bioreactors: effect of q power input and medium viscosity * Arindom Sen, Michael S. Kallos, Leo A. Behie Pharmaceutical Production Research Facility ( PPRF), Faculty of Engineering, University of Calgary, 2500 University Dr. N. W ., Calgary, Alberta, Canada T2N 1N4 Accepted 10 December 2001 Abstract 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 ( D ,150 mm) below sizes MAVG 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 2 cell damage ( t 5 9.76 dyn / cm ). This is a first step in the development of a reproducible, scaled-up process for the production of max neural stem cells for therapeutic applications including the treatment of neurodegenerative disorders and acute central nervous system injuries.  2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Regeneration Keywords: Neural stem cell; Expansion; Cell aggregate; Suspension bioreactor; Shear; Medium viscosity; Power input 1. Introduction mal growth factor (EGF) and fibroblast growth factor (bFGF) [5,14,15,18]. It has been shown that the neural 1.1. Neural stem cells stem cells (NSC) can be cultured for extended periods of time (up to ten passages) in stationary culture without Multipotent neural precursor cells can be isolated from losing their pluripotency [15]. We have previously ex- the adult and embryonic murine central nervous system amined the serial cultivation of NSC in suspension culture (CNS) and induced through epigenetic means to proliferate (eight passages) to test whether or not these cells respond in vitro [14,15]. These non-transformed mammalian neural favorably to shear, and/or change their phenotype in cells form spherical aggregates of stem cells and response to agitation [4]. It was found that it was possible 7 progenitor cells when cultured on non-adherent tissue to obtain large expansion ratios (10 in 35 days) of murine culture plastic in the presence of mitogens such as epider- NSC in suspension bioreactors without the loss of pluripotency. Transfer of nutrients to cells within the NSC aggregates q This manuscript was presented at the 4th Brain Research Interactive from the bulk medium and transfer of metabolic by- Symposium—Stem Cells in the Mammalian Brain in San Diego, CA, products from these cells to the bulk medium is achieved USA, November. 8–10, 2001. through diffusion. As such, nutrient and metabolic by- *Corresponding author. Tel.: 11-403-220-6692; fax: 11-403-284- product concentration profiles exist within an aggregate 4852. E-mail address: behie@ucalgary.ca (L.A. Behie). with nutrient concentration levels being greatest at the 0165-3806 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0165-3806(01)00328-5