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Keywords:

  • shear stress;
  • rheology;
  • concentration;
  • bioprocessing;
  • tissue engineering;
  • cell therapy;
  • mammalian cell;
  • centrifugation;
  • cytometry

Abstract

Regenerative medicines based on human cells demand their harvesting, culture, and processing. Manufacturing processes are likely to include cell concentration and subsequent controlled dosing of concentrates, for example, to the patient or tissue construct. The integrity and functionality of the cells must be maintained during these processing stages. In this study the performance of two different cell concentration protocols (involving centrifugation and resuspension) are compared and consideration given to possible causes of cell loss. Further studies examine cell size and rheological behavior of anchorage-dependent mammalian cell suspensions, and the effect of capillary flow stress (0.5–15 Pa, laminar flow regime) on cell number and membrane integrity as quantified by flow cytometry. The cell concentration protocols achieved maximum cell volume fraction of around 0.3 and the improved protocol exhibited intact cell yield of 80 ± 13%, demonstrating proof-of principle for achieving tissue-like cell concentrations by a process of centrifugation and orbital shaking. Volume mean cell diameter (cell diameter at the mean cell volume) for the rat aortic smooth muscle cells (CRL-1444) used in this study was 22.4 µm. Concentrated cell suspension rheology approximated to power law behavior and exhibited similar trends to reports for plant and yeast cells. Capillary transfer at 2–15 Pa (wall shear stress) did not significantly affect cell number or membrane integrity while losses observed at low shear (0.5, 1.0 Pa) were probably due to surface attachment of cells in the apparatus. Biotechnol. Bioeng. 2009;103: 1236–1247. © 2009 Wiley Periodicals, Inc.