title: Two parametric cell cycle analyses of plant cell suspension cultures with fragile, isolated nuclei to investigate heterogeneity in growth of batch cultivations.
abstract: Plant cell suspensions are frequently considered to be heterogeneous with respect to growth in terms of progression of the cells through the cell cycle and biomass accumulation. Thus, segregated data of fractions in different cycle phases during cultivation is needed to develop robust production processes. Bromodeoxyuridine (BrdU) incorporation and BrdU-antibodies or 5-ethynyl-2'-deoxyuridine (EdU) click-it chemistry are frequently used to acquire such information. However, their use requires centrifugation steps that cannot be readily applied to sensitive cells, particularly if nuclei have to be extracted from the protective cellular milieu and envelopes for DNA analysis. Therefore, we have established a BrdU-Hoechst stain quenching protocol for analyzing nuclei directly isolated from delicate plant cell suspension cultures. After adding BrdU to test Harpagophytum procumbens cell suspension cultures the cell cycle distribution could be adequately resolved using its incorporation for the following 72 h (after which BrdU slowed biomass accumulation). Despite this limitation, the protocol allows resolution of the cell cycle distribution of cultures that cannot be analyzed using commonly applied methods due to the cells' fragility. The presented protocol enabled analysis of cycling heterogeneities in H. procumbens batch cultivations, and thus should facilitate process control of secondary metabolite production from fragile plant in vitro cultures. Biotechnol. Bioeng. 2016;113: 1244-1250. © 2015 Wiley Periodicals, Inc.
authors.affiliation: Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Bergstraße 120, Dresden, 01062, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Haas
authors.affiliation: Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Bergstraße 120, Dresden, 01062, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Hegner
authors.affiliation: Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Bergstraße 120, Dresden, 01062, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Helbig
authors.affiliation: Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Bergstraße 120, Dresden, 01062, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Bartels
authors.affiliation: Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Bergstraße 120, Dresden, 01062, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bley
authors.affiliation: Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Bergstraße 120, Dresden, 01062, Germany. jost.weber@tu-dresden.de.
authors.forename: Jost
authors.initials: J
authors.lastname: Weber
mesh_terms: D061249:Batch Cell Culture Techniques / Q000379:methods*; D002453:Cell Cycle / Q000502:physiology; D002467:Cell Nucleus / Q000502:physiology* / Q000648:ultrastructure; D049109:Cell Proliferation / Q000502:physiology*; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D031684:Harpagophytum / Q000166:cytology* / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000379:methods
keywords: Harpagophytum procumbens; Hoechst-BrdU-quenching; plant cell suspension and in vitro culture; process monitoring; proliferation activity; two-parametric cell cycle analysis