Plant Cell Culture Monitoring Using an in Situ Multiwavelength Fluorescence Probe Steve Hisiger and Mario Jolicoeur* Canada Research Chair on the Development of Metabolic Engineering Tools, Bio-P 2 , Department of Chemical Engineering, E Ä cole Polytechnique de Montre ´al, P.O. Box 6079 Centre-ville Station, Montre ´al, Que ´bec, Canada A multiwavelength fluorescence probe is proposed for in situ monitoring of Eschscholt- zia californica and Catharanthus roseus plant cell cultures. The potential of the probe as a tool for real-time estimation of biomass and production in secondary metabolites has been studied. The probe excitation range is 270-550 nm and the emission range is 310-590 nm, with a step of 20 nm for both excitation and emission filters. Many endogenous fluorophores such as NAD(P)H, riboflavins (riboflavin and derivatives such as FMN, FAD), tryptamine and tryptophan, and fluorescent secondary metabolites were analyzed simultaneously. NAD(P)H fluorescence signal (350/450 nm) showed to be an adequate signal for estimating cells activity. Riboflavins fluorescence signal (450/ 530 nm) followed C. roseus cell concentration both for the growth phase and after elicitation with jasmonic acid. Fluorescence from the alkaloids interfered with NAD- (P)H signal during the production phase. For C. roseus, tryptophan, tryptamine, ajmalicine and serpentine were monitored by the probe. For E. californica, fluorescence from alkaloids overlapped with riboflavins preventing from using the probe to follow cell growth but global alkaloids production could be followed using the probe. Introduction Fluorescence measurement is a very sensitive method enabling noninvasive detection at a micromolar level. In the past decades, several fluorescence probes that can be used in situ in bioreactors (Hitachi, Ocean optics, Delta optics, Ingold) were developed to monitor culture behavior. Most of these probes were limited to monitoring a unique parameter using predetermined excitation/ emission (ex/em) wavelengths. Indirect biomass estima- tion was performed through different autofluorescent intracellular molecules such as tryptophan and NAD- (P)H. Furthermore, monitoring of the degradation or the production of a fluorescent compound has also been reported (1, 2). However, the development of multiwave- length probes allowing scanning cell suspensions at different excitation and emission wavelengths has sig- nificantly increased the amount and quality of the information to be monitored. One can then target several specific fluorescent molecules through the use of specific optic filters mounted on filterwheels. The time delay between each fluorescence signal measurement then depends on mechanical and electronic delays required to change excitation and emission filters. Biomass and redox potential have been estimated on-line via fluorescence signals from tryptophan and NAD(P)H, respectively, using a single probe (3). Tartakovsky et al. (4) were able to estimate growth, glucose and metabolic byproducts production while monitoring E. coli and S. cerevisae cultures. These authors scanned a 2D fluorescence spectrum and analyzed by a stepwise multiple regression procedure selecting specific pairs (excitation/emission) of wavelengths. They found that tryptophan, pyridoxine and riboflavins signal could be correlated with biomass concentration, and signal from an undetermined com- pound (270/500 nm) allowed for the estimation of glucose concentration. Similar study performed with Pseudomo- nas fluorescens (5) revealed that O 2 in the exhaust gas could be followed via the integration of multiple signals above 290/390 nm. For quantitative studies using mo- lecular probes such as for intracellular pH and Ca 2+ , ratiometric measurements using dual excitation and emission wavelengths have shown to be more reliable than using unique florescence signal (6 and references therein). Most plant phytomolecules of therapeutic interest are composed of several aromatic rings with delocalized π electrons. These molecules can thus be detected under fluorescence and/or UV absorbance. Phytomolecules iden- tification and quantification can be performed using HPLC and TLC techniques. Several extraction and concentration steps are usually needed before performing HPLC and TLC analysis. In addition, reproducible and representative sampling of plant cell suspension in a bioreactor is tedious due to plant cell suspension char- acteristics. Plant cell suspensions are highly viscous, show rheofluidifiant behavior at high density (7) and exhibit a large distribution of cell and aggregate sizes. Therefore, the use of a probe capable of on-line estimation of culture state parameters such as cell concentration, cellular activity level and secondary metabolites produc- tion could then be highly useful. In this work the use of a multiwavelength fluorescence probe was studied to monitor plant cell culture. The scope of this study was to assess the probe capacity to follow directly on-line plant cell concentration, cellular activity, precursors of the secondary metabolism and secondary metabolites. Suspension cell cultures were performed with Eschscholtzia californica and Catharanthus roseus in shake flasks and in bioreactor, respectively. * To whom correspondence should be addressed. Ph: 1-514-340- 4711ext.4525.Fax: 1-514-340-4159.E-mail: mario.jolicoeur@polymtl.ca. 580 Biotechnol. Prog. 2005, 21, 580-589 10.1021/bp049726f CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical Engineers Published on Web 02/10/2005