Enhancement of Sf-9 Cell Growth and Longevity through Supplementation of Culture Medium with Hemolymph Luis Maranga, †,⊥ Ronaldo Z. Mendonc ¸ a, ‡,⊥ Andre ´ Bengala, † Cristina C. Peixoto, † Roberto H. P. Moraes, § Carlos A. Pereira, ‡ and Manuel J. T. Carrondo* ,†,| Instituto de Biologia Experimental e Tecnolo ´gica/Instituto de Tecnologia Quı ´mica e Biolo ´gica IBET/ITQB, Apartado 12, P-2781-901 Oeiras, Portugal; Laborato ´rio de Imunologia Viral and Laborato ´rio de Parasitologia, Instituto Butantan, Av. Vital Brasil 1500 CEP. 05503-900 Sa ˜ o Paulo SP, Brasil; and Laborato ´rio de Engenharia Bioquı ´mica, Faculdade de Cie ˆncias e Tecnologia, Universidade Nova de Lisboa, P-2825-114 Monte da Caparica, Portugal The benefits of insect cell culture medium supplementation with hemolymph of Lonomia obliqua were investigated. The addition of hemolymph to the medium induced high levels of cell growth, and the viability was maintained for longer periods. The maximum cell yield increased almost 3-fold after hemolymph supplementation. Cultures in their stationary phase were rescued through hemolymph supplementation, also reaching high cell concentrations. These actions were much dependent on the concentration of hemolymph; low hemolymph concentration had a positive effect in cell growth, whereas high hemolymph concentration showed a deleterious effect. Fractionation of hemolymph by gel filtration chromatography showed the presence of three factors with different activity in insect cell culture: an potential anti-apoptotic factor, a growth-promoting factor, and an enzyme that hydrolyzes sucrose. Addition of hemolymph to the medium induced high levels of glucose production. The sucrose to glucose conversion was also linearly dependent upon the hemolymph concentration. Therefore, we conclude that cell growth and longevity can be increased by supple- mentation of the culture medium with hemolymph. 1. Introduction The insect cell-baculovirus expression system has been broadly used to produce all types of recombinant proteins (1). The early development of optimal culture conditions for the establishment of continuous insect cell lines involved the use of media supplemented with hemolymph (2-5) and Grace’s medium, and the first successful formulation for developing cell lines was actually based on the composition of insect hemolymph. However, because hemolymph was difficult to obtain in generous amounts, in the 1960s and 1970s it was successfully replaced with fetal bovine serum (FBS), whole egg ultrafiltrate, turkey serum, or even chicken serum (6- 8). The adaptation of FBS for use with mammalian cell cultures and the resulting commercial availability led to this supplement becoming standard in insect cell culture (9). Several studies have shown the presence of compounds with pharmacological activity in the hemolymph of some insects: either enzymatic (10-15), hormonal (16-20), immunoreactant, antibacterial or fungicidal (21-26), or antifreeze compounds (27). Understanding the beneficial properties of the hemo- lymph could allow us to design less expensive and better- characterized serum-free culture media. Hemolymph could also be the source of some yet unidentified proteins acting directly in the modulation of some cellular function with potential biotechnological application. Recently Rhee et al. (28) and Rhee and Park (29) showed that silkworm hemolymph has a positive effect on cell longevity after baculovirus infection by delaying cell death. Those authors suggested that silkworm hemolymph inhibits baculovirus-induced insect cell apop- tosis. Later Kim et al. (30) isolated and characterized the apoptosis-inhibitor component present on hemolymph of Bombyx mori responsible for this effect. Kanaya and Kobayashi (31) have also reported the purification and characterization of a 15.2-kDa insect hemolymph protein that promotes the in vitro replication of the B. mori nucleopolyhedrovirus (BmMNPV), also enhancing the production and activity of a recombinant protein by about 6,000 times. Earlier, Woo et al. (32) also demonstrated that hemolymph of B.mori added to the medium accelerated the expression of -galactosidase about two times, and this effect was both cell and hemolymph concentration dependent. However, silkworm (B. mori) hemolymph has been mainly studied regarding baculovirus infection. Overall, data about the effect of hemolymph in cell growth is scarce. Lonomia obliqua (Leptidoptera:Saturniidae) is an in- sect of medical importance due to the presence of poison spines in their larvae (33). A specific antiserum for the poison, developed in horses, was produced at Instituto Butantan (Sa ˜ o Paulo, Brazil), and its use successfully * To whom all correspondence should be addressed at IBET. Ph: ++351 21 442 77 87. Fax: ++351 21 442 11 61. E-mail: mjtc@ itqb.unl.pt. † IBET/ITQB. ‡ Laborato ´rio de Imunologia Viral, Instituto Butantan. § Laborato ´rio de Parasitologia, Instituto Butantan. | Universidade Nova de Lisboa. ⊥ Both authors contributed equally to the preparation of this manuscript. 58 Biotechnol. Prog. 2003, 19, 58-63 10.1021/bp025583q CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 11/28/2002