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Flow Cytometric Identification of Paclitaxel‐Accumulating Subpopulations
Author(s) -
Naill Michael C.,
Roberts Susan C.
Publication year - 2008
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp049544l
Subject(s) - paclitaxel , flow cytometry , cell sorting , cell culture , cell , methyl jasmonate , intracellular , biology , chemistry , microbiology and biotechnology , biochemistry , chemotherapy , genetics , gene
An immunofluorescence procedure was developed for paclitaxel quantification at the single cell level via flow cytometry in Taxus cuspidata suspension cultures. Intracellular staining was validated via fluorescence microscopy. Paclitaxel content of isolated cells and protoplasts was compared to total paclitaxel levels measured via HPLC. Paclitaxel accumulation was significantly increased by elicitation with methyl jasmonate (100 μM) on day 7 post‐transfer as compared to unelicited cultures. Maximum accumulation was observed by day 12 post‐transfer in both total paclitaxel (∼0.25 mg/L) and the percentage of paclitaxel‐accumulating cells (∼95%). A similar trend was observed with isolated protoplasts, although protoplasts accumulated only ca. 40–75% of the paclitaxel present in single cells. In unelicited cell cultures, a small subpopulation (ca. 3–5%) of single cells was shown to accumulate paclitaxel. Although nearly all cells were observed to accumulate paclitaxel in methyl jasmonate‐elicited cell cultures, a high degree of cell‐to‐cell variation was observed in paclitaxel content. The identified subpopulations represent targets for cell sorting, which may be applied to develop higher‐accumulating cell lines. The quantification of single cell paclitaxel content is useful for characterizing production variability in cell cultures and can be utilized to develop rational strategies to increase paclitaxel production.