Growth, Nutrient Consumption, and End‐Product Accumulation in Sf‐9 and BTI‐EAA Insect Cell Cultures: Insights into Growth Limitation and Metabolism

Abstract

Growth, nutrient consumption, and end-product accumulation were quantitated in shake-flask cultures of two insect cell lines, Sf-9 and BTI-EAA, in three different serum-supplemented media. Per cell consumption or production rates were calculated for most medium components analyzed. Glucose was growth-limiting in TNM-FH medium and was the most important single source of organic-C for the cells in all cultures. Cells utilized fructose and maltose but not sucrose. alpha-Ketoglutarate and malate contributed significantly to the carbon budget of cells in TNM-FH. Lactate generally did not accumulate during growth. Most of the amino acids were consumed by the cells, with the exception of alanine which was produced. Most of the amino acids appeared to be present in adequate supply in the cultures. Glutamate was generally the most rapidly consumed of the amino acids, followed closely by glutamine. Alanine accumulation was correlated with glucose consumption. In Sf-9 cultures, ammonia accumulated only slightly or not at all as long as glucose was present in the medium, and uric acid was detectable at the end of growth and in the stationary phase. Added ammonia up to a concentration of 10 mM did not affect the growth of either cell line. Ammonia and lactate may be of less importance in limiting growth in insect cell cultures than in mammalian cell cultures. A hypothetical outline of the major metabolic pathways of the cultured insect cells is presented on the basis of information obtained here and in the literature.

Growth, metabolism and baculovirus production in suspension cultures of an Anticarsia gemmatalis cell line

“…The maximum value of 5.87 · 10 -14 mol cell -1 h -1 , reached after 48 h in culture, was higher than that measured in other lepidopteran insect cell lines (Bé dard et al 1993;Rhiel et al 1997;Mendonça et al 1999). This high consumption rate of glutamine could also be related to the low glucose concentration in the TC-100 medium, taking into account the role of glutamine as an alternative energy source to carbohydrates in insect cell cultures.…”

“…Glucose, the unique carbohydrate present in TC-100 medium, was the most widely consumed nutrient in saU-FL-AG-286 cultures. The cell-specific glucose consumption rate in saUFL-AG-286 cultures was slightly lower than the maximum rates reported in cultures of other insect cell lines under similar culture conditions (Bé dard et al 1993;Drews et al 1995;Mendonça et al 1999;Rhiel et al 1997). This low rate of glucose consumption could be due to the low concentration of glucose in TC-100 medium.…”

“…The oversupply of amino acids by insect cell culture media with respect to the cellular requirements is also a fact reported in cultures of Spodoptera frugiperda cell lines (Bé dard et al 1993;Ferrance et al 1993). It should be noted that the concentration of cysteine, an amino acid highly consumed by Sf9 cells and whose concentration determines the uptake rate of other amino acids (Doverskog et al 1998), has not been analyzed in this work.…”

“…The accumulation of by-products could also be the cause of viability reduction. Ammonia is a toxic metabolite in animal cell cultures, but the concentration reached in saUFL-AG-286 cultures should not be considered detrimental for culture viability, according to results obtained with other insect cell lines (Bé dard et al 1993;Ö hman et al 1996). Lactate, another by-product commonly produced in animal cell cultures, did not accumulate in saUFL-AG-286 culture supernatants.…”

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

“…Since glucose was still available in Sf900 II medium when lactate concentration increased, oxygen limitation probably occurred in this particular situation, as previously discussed by Mendonc¸a et al (1999). Upon glucose limitation, the lactate accumulated in Grace's medium (control) and in formulation 3 medium was consumed simultaneously to this carbon source, as previously reported by other groups (Bedard et al 1993;Palomares and Ramı´rez 1996;Mendonc¸a et al 1999). Interestingly, total glutamine consumption was lower than 0.31 g l À1 in both formulation 3 and in Grace's control medium, however, it reached around 1 g l À1 in Sf900 II medium (data not shown).…”