-
It is well known that insect cell lines have been used for the study of insect and plant viruses [6, 8]. In particular, with the advent of the baculovirus-insect cell system, a new exciting application for insect cells in biotechnology was realized to provide adequate levels of recombinant proteins or maximum production of viruses. The optimum growth temperatures for different cells are an important factor [3, 8, 15]. Usually the optimal growth temperature of insect cell lines ranges from 25℃ to 33℃ with an optimum at 28℃[7]. A temperature shift to 45℃ leads to the death of Drosophila cells, except when cells have been pretreated to an intermediate temperature for a short time, i.e, 1h at 37℃[7, 16]. Survival at a stress temperature has been correlated with the synthesis of heat shock proteins (hsp) first described in Drosophila [4, 17], and further documented in many prokaryotic and eukaryotic cells subjected to a transient rise in temperature [1, 2, 13]. Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells[5, 11]. They have a distinct karyotype, membrane lipid composition, morphology, and growth kinetics from the parental cell lines. Also Lynn reported that virus appeared to have the highest titer in some cells when at 22℃ but the time required to reach the maximum titer was slower at this temperature[14].
In this paper, we describe the development of ht cell lines, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1), from other insect species Trichoplusia ni [9]. Adaptation of cell lines High5 and MG1 to 33℃ and 35℃ was carried out by shifting the culture temperature between 28℃ and higher temperatures by a gradual stepwise increase in temperature. The cell-lines with the temperature adaptation were designated as sf9-ht33, sf9-ht35, High5-ht33, High5-ht35, MG1-ht33, and MG1-ht35. Then we examined their charac-teristics in different temperatures.
HTML
-
Cell morphology
Cell lines sf-9, High5, MG1, and their heat adapted to 33℃ (ht33) and 35℃ (ht35) counterparts were observed by microscope. Each image of the cultures was captured at 3 days after incubation at their adapted temperatures (Fig. 1). All of cell lines grew as anchorage dependent cells in a T-flask. The morphology of the cells, grown in TNM-FH medium, did not obviously change, however the cell size of sf9-ht cells was enlarged and those of High5 and MG1 cells were reduced after heat adaptation (Table. 1).
Figure 1. Microscopic observation of cell lines sf9, High5, MG1 (parental) and their sub-cell lines heat adapted to 33℃ (ht33) and 35℃ (ht35). Image of the culture was captured at 3 days after incubation at their adapted temperatures. An internal scale bar represents 50 μm
Table 1. Characteristics of heat tolerant cell lines derived from Sf9, High5 and MG1
Growth of cell lines
Growth curves of cultured cells of sf-9, High5, MG1, and their heat adapted cells were determined at their adapted temperatures (Fig. 2). All heat adapted cell lines were faster-growing than that those of parental cell lines. The population doubling times of sf9-ht35, High5-ht35 and MG1-ht35 cells were 4, 3 and 1 h less than those of their parental cell lines, respectively (Table. 1), and the times that cells reached a maximal density were shortened with the rise of culture temperature (Fig. 2).
Figure 2. Growth curves of cultured cells of sf9, sf9-ht33, sf9-ht35(A), High5, High5-ht33, High5-ht35(B), MG1, MG1-ht33, MG1-ht35 (C) at their adapted temperatures, 28℃, 33℃ and 35℃, respectively. Point of ○, □ or △ and length of the vertical line represents a mean and standard deviation of the measured cell densities
Viral susceptibility and production
The susceptibility of both parental cells (sf-9, High5, and MG1) and their ht cells (sf9-ht33, sf9-ht35, High5-ht33, High5-ht35, MG1-ht33, MG1-ht35) to wild type AcMNPV at their adapted temperatures was approximately similar with a infection of over 95% of the cells 3 days P. i. (Fig. 3).
Figure 3. Microscopic observation of cell lines sf9, High5, MG1 (parental) and their ht cell lines infected with AcMNPV at their adapted temperatures. Image of the infected culture was captured at 4 days post-infection. interval scale bar represents 50 μm
The production of budded virus (BV) and occlusion body (OB) by these cell lines at different temperatures was measured 3 days P. i. and was optimum at its own adapted temperature (Fig. 4). When at 28℃ culture, the parental cells, sf-9, High5, and MG1, showed the highest level of BV production (average of 7.8×107, 5.7×107 and 5.2×107 TCID50/mL, respectively) in comparison to the lower level produced in ht-33 (average of 3.0×107, 5.0×107 and 4.4×107 TCID50/mL, respectively) and ht-35 cells (average of 2.5×107, 2.2×107 and 7.0×106 TCID50/mL, respectively) (Fig. 4). When at 33℃ culture, the production of BV by the ht-33 cells, sf9-ht33, High5-ht33, and MG1-ht33, was the highest (average of 3.5×107, 3.7×107 and 4.6×107 TCID50/mL, respectively) as compared to parental cells and ht-35 cells. When at 35℃ culture, all of the ht-35 cells produced over sevenfold more BV than parental cells and ht-33 cells. The production of occlusion body by these cells showed a similar trend to the results with BV production.
Figure 4. AcMNPV production of sf9, High5, MG1(A), sf9-h33, High5-ht33, MG1-ht33(B), sf9-ht35, High5-ht35, MG1-ht35 (C) at 28℃, 33℃, 35℃ and 37℃. At 4 days after infection, culture medium and cells containing AcMNPV BV and OB were collected and measured for their virus production. Length of bars and lines on top of their represent means standard deviation of the production, respectively
Changes of cell density in cultures of their adapted temperatures
The serial passage of parental cells and their ht cells during subculture at 28℃, 33℃ and 35℃ demonstrated that each cell line had an constant density at its own adapted temperature through the ninth passage (Fig. 5).
Figure 5. Change of cell density in cultures of sf9, High5, MG1 (parental) and their ht cell lines during subcultures at 28℃, 33℃ and 35℃. Point of ○, □ and △ represent a mean of the measured cell densities
At 28℃ culture, the parental cells (sf-9 and High5) were able to keep an almost constant density between 1.5×106and 2.0×106cells/mL, but the sf9-ht33 and High5-ht35 had not adapted to the lower temperature, so the cell density in cultures of both sf9-ht and High5-ht cells went rapidly down after 3ird passage. However, all of MG1 cells including ht-33 and ht-35 could suit this low temperature with higher cell density (between 1.6×106 and 1.8×106 cells/mL) in comparison to sf-9 and High5 cells. At 33℃ culture, the parental cell lines of sf-9 and High5 could not adapt to the higher temperature and the cell density gradually decreased with the increase of subculture passage. But other ht cells including MG1 parental cells could always keep higher cell density. At 35℃ culture, all of the ht35 cells including sf9-ht35, High5-ht35, and MG1-ht35 were able to still maintain the highest level of cell density in comparison to other cells investigated in this study.