Monoterpenoid Oxindole Alkaloid Production by Uncaria tomentosa (Willd) D.C. Cell Suspension Cultures in a Stirred Tank Bioreactor Gabriela Trejo-Tapia, †,‡ Carlos M. Cerda-Garcı ´a-Rojas, § Mario Rodrı ´guez-Monroy, ‡ and Ana C. Ramos-Valdivia* ,† Departamento de Biotecnologı ´a y Bioingenierı ´a, Centro de Investigacio ´n y de Estudios Avanzados del IPN (CINVESTAV-IPN), Av. Instituto Polite ´cnico Nacional 2508, Col. San Pedro Zacatenco, 07360, Me ´xico, D. F., Mexico, Departamento de Biotecnologı ´a, Centro de Desarrollo de Productos Bio ´ticos del IPN (CEPROBI-IPN), P.O. Box 24, 62730, Yautepec, Morelos, Mexico, and Departamento de Quı ´mica, Centro de Investigacio ´n y de Estudios Avanzados del IPN (CINVESTAV-IPN), Av. Instituto Polite ´cnico Nacional 2508, Col. San Pedro Zacatenco, 07360, Me ´xico, D. F., Mexico Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 ( 0.6 g DW/L and a specific growth rate of 0.102 d -1 . U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 ( 40.0 μg/L, 44.6 ( 5.2 μg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor. Introduction Uncaria tomentosa (Willd) D.C. (Rubiaceae), commonly known as “un ˜ a de gato” or “cat’s claw”, is a large woody vine indigenous to the Amazon rainforests of Peru, Brazil, and Ecuador. This plant is widely used in traditional Peruvian medicine (1). U. tomentosa contains a diverse array of compounds, including sterols, triterpenes, fla- vonoids, and tetra- and pentacyclic monoterpenoid oxin- dole alkaloids (MOA) (2-4). Among these secondary metabolites, pentacyclic MOA are the most attractive substances because of their immunomodulatory (4, 5), cytotoxic, anti-AIDS, and antileukemic activities (3-5). Due to the important pharmacological properties of MOA, U. tomentosa is widely commercialized as powdered bark. This material is obtained from plants of at least 8 years old growing in the wild (1, 4), whose average natural occurrence is five specimens per hectare in some regions of the Amazon rainforest (4). Several problems are associated with this extraction method as quantities and qualities of the plant material are variable (6). Addition- ally, given the structural complexity of MOA, chemical synthesis of these compounds is quite challenging and presents many disadvantages such as the generation of stereoisomeric mixtures. Hence, plant cell culture might represent a biotechnological alternative to produce these demanded and valuable compounds. The exploitation of in vitro culture techniques requires high-producing cell lines and large-scale cell cultivation in bioreactors. The excretion of the product to the culture medium is also desirable (6). The plant cell growth environment in a bioreactor is different from that in Erlenmeyer’s flasks; changes in shear stress, oxygen supply and gas composition are expected (6-8). In particular, plant cells have generally been described as sensitive to shear stress, impeding the culture of many cell lines in bioreactors. Meijer et al. (9) reported that suspension cultures of Cinchona robusta (Rubiaceae) were not able to grow in a stirred tank. Nevertheless, several studies point out that the susceptibility of plant cells to shear stress varies greatly with cell lines (9, 10). Cell suspension cultures grown in bioreactors are exposed to higher hydrodynamic stress in comparison to those in Erlenmeyer flasks (8). Jua ´ rez et al. (11) reported the values of power input as an indicator of hydrody- namic stress intensity for Erlenmeyer flasks and a 2-L laboratory stirred tank. Power input for this stirred tank was 1 order of magnitude higher than that of Erlenmeyer flasks. Some characteristics of the plant cell cultures developed in bioreactors have been associated with the damage caused by the hydrodynamic stress. These in- clude alteration in single cells and aggregate size (12) and shape (13), loss of viability, lower cell growth, and/ * To whom correspondence should be addressed. Ph: +52 55 50 61 38 00 ext. 4327. Fax: +52 55 50 61 33 13. E-mail: aramos@cinvestav.mx. † Departamento de Biotecnologı ´a y Bioingenierı ´a, CINVESTAV- IPN. ‡ Departamento de Biotecnologı ´a, CEPROBI-IPN. § Departamento de Quı ´mica, CINVESTAV-IPN. 786 Biotechnol. Prog. 2005, 21, 786-792 10.1021/bp049608s CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical Engineers Published on Web 03/19/2005