Quantitative Studies of Cell-Bubble Interactions and Cell Damage at Different Pluronic F-68 and Cell Concentrations

Authors

  • Ningning Ma,

    1. Fermentation and Cell Culture, Bioprocess R & D, Merck Research Laboratories, Merck & Co., Inc., West Point, PA, 19486
    2. Department of Chemical Engineering, The Ohio State University, Columbus, Ohio 43210
    Current affiliation:
    1. Pfizer PGRD, St. Louis, MO 63167
    Search for more papers by this author
  • Jeffery J. Chalmers,

    1. Department of Chemical Engineering, The Ohio State University, Columbus, Ohio 43210
    Search for more papers by this author
  • John G. Auniņš,

    Corresponding author
    1. Fermentation and Cell Culture, Bioprocess R & D, Merck Research Laboratories, Merck & Co., Inc., West Point, PA, 19486
    • Fermentation and Cell Culture, Bioprocess R & D, Merck Research Laboratories, Merck & Co., Inc., West Point, PA, 19486. Tel: 215–652–5577. Fax: 215–993–4880
    Search for more papers by this author
  • Weichang Zhou,

    1. Fermentation and Cell Culture, Bioprocess R & D, Merck Research Laboratories, Merck & Co., Inc., West Point, PA, 19486
    Current affiliation:
    1. Protein Design Labs, Fremont, CA 94555
    Search for more papers by this author
  • Liangzhi Xie

    1. Fermentation and Cell Culture, Bioprocess R & D, Merck Research Laboratories, Merck & Co., Inc., West Point, PA, 19486
    Current affiliation:
    1. Cell Culture R&D Center, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, P. R. China
    Search for more papers by this author

Abstract

Pluronic F-68 (PF-68) is routinely used as a shear-protection additive in mammalian cell cultures. However, most previous studies of its shear protection mechanisms have typically been qualitative in nature and have not covered a wide range of PF-68 and cell concentrations. In this study, interactions between air bubbles along with the associated cell damage were investigated using the novel adenovirus-producing cell line PER.C6, a human embryonic retinoblast transfected with the adenovirus type 5 E1 gene. A wide range of PF-68 and cell concentrations (approximately 3 orders of magnitude) were used in these studies. At low PF-68 concentrations (0.001 g/L), cells had a very high affinity for bubbles, indicated by a more than 10-fold increase in cell concentration in the foam layer liquid versus the bulk liquid. At high PF-68 concentrations (∼3 g/L), however, the cell concentration in the foam layer liquid was only ∼40% of that in the bulk cell suspension. The number of cells associated with each bubble decreased from approximately 1000 cells at 0.001 g/L PF-68 to approximately 120 cells at 3 g/L PF-68. Despite the lower cell affinity for bubbles at a high PF-68 concentration, at high cell concentrations (107 cells/mL and 1 g/L PF-68) significant cell entrapment occurred in the foam layer, on the order of 1000 cells/bubble. For the cells carried by the bubbles, quantitative cell damage data revealed that the probability of cell death from bubble rupture was independent of bulk cell concentration but was affected by PF-68 concentration. These quantitative studies further indicated that even at a low PF-68 concentration of 0.03 g/L, approximately 30% of the attached cells were killed during the bubble rupture process. At the same time, at low PF-68 concentration (<0.1 g/L), significant cell death occurred prior to bubble rupture. On average, a bubble disrupted more cells in the bulk liquid and/or foam layer than during rupture. For both mechanisms, the number of cells damaged by each bubble increased with decreasing PF-68 concentration and increasing bulk cell concentration.

Ancillary