Effect of Temperature Oscillation on Insect Cell Growth and Baculovirus Replication

Abstract

Temperature oscillation can enhance cell viability of sf9 insect cells and baculovirus production of occlusion bodies (OB) and extracellular virus (ECV) compared with constant temperature in stationary culture and suspension culture. The optimal oscillation range was 24 to 28°C. At this temperature oscillation, the viability of uninfected and infected sf9 cells can be maintained much longer than at 28°C. Although the rate of virus infection was a little low at 24 to 28°C, the final cell infectivity was similar to that at a constant temperature of 28°C. The production of OB was increased from 13.4 to 17.4/cell in stationary culture and from 13.9/cell to 18.1/cell in suspension culture. The titer of ECV was increased from 87 to 114 PFU/cell in stationary culture and from 79 to 114 PFU/cell in suspension culture.

Insect cell culture is an emerging technology for the production of biologicals, including recombinant proteins and biological insecticides. The optimization of insect cell culture systems for the production of insect viruses, primarily those from the Baculoviridae family, has been the focus of many studies (2). One of the key points is how to enhance the insect cell growth and baculovirus production through the improvement of culture conditions. Temperature is an important factor affecting insect cell growth and baculovirus replication (4). The temperature range of 25 to 30°C is favorable to insect cell growth, and the optimal range is 27 to 28°C (1). However, the number of occlusion bodies (OB) produced per cell increased at 25 and 32°C compared with 28°C (3). This shows that the optimal temperature for cell growth is not necessarily the same as that for the production of virus. Most present studies of insect cell culture use a constant temperature in the culture process. This work reports a novel operation in the culture process, temperature oscillation, and studies its effect on insect cell growth and baculovirus replication.

MATERIALS AND METHODS

Cell line and virus stock.

The sf9 insect cell line (Spodoptera frugiperda) was provided by the College of Life Sciences, Peking University. Cells were grown in TC-100 medium (Gibco) supplemented with NaHCO₃ (0.5 g/liter)–10% (vol/vol) new-born calf serum (Beijing, People’s Republic of China).

The wild baculovirus Autographa californica nuclear polyhedrosis virus was provided by Li Guoxun (East-North Agriculture University, Haerbin, People’s Republic of China).

Culture conditions and assay.

For stationary cultures, 25-cm² tissue culture flasks (Nunclon) with 5 ml of medium were used and the seeding density was 2.8 × 10⁵ cells/ml. For suspension cultures, 150-ml spinner flasks, each with a 50-ml working volume, were used and the seeding density was 3.0 × 10⁵ cells/ml. The agitation rate was set at 80 rpm. Cell density was determined with a hemocytometer, and viability was assessed by Trypan Blue (0.2%) exclusion.

Cells were inoculated with baculovirus at a multiplicity of infection of 0.5 on the 3rd day of culture. Virus was harvested 4 days postinfection. Total virus production, including the titer of extracellular virus (ECV) and the number of OB, was determined by end point assay (9) and counting with a hemocyometer, respectively. The percentage of infected cells was obtained by microscopic counting of cells with polyhedral inclusion bodies.

The counts of OB and cells were replicated five times in each experiment.

Temperature oscillation.

Cells were grown at 28°C for 2 days and then transferred to different temperature oscillation conditions. The upper temperature limit was kept at 28°C, and the lower limit was 20, 22, 24, or 26°C. Based on preparatory experiments, the switching interval of oscillation was set at 12 h. The culture at a constant temperature of 28°C was used as a control for all comparisons made in this study since 28°C was optimal for baculovirus production among four constant temperatures: 22, 24, 26, and 28°C (data not shown).

RESULTS

Effect of temperature oscillation on uninfected insect cell growth.

Figure 1 shows the growth of uninfected insect cells in stationary cultures with different temperature oscillations. The 28°C culture, the culture oscillating between 26 and 28°C, and the culture oscillating between 24 and 28°C had approximately the same maximal viable cell densities (18.4 × 10⁵, 18.8 × 10⁵, and 18.9 × 10⁵ cells/ml, respectively). The maximal cell density was reached at the 4th day in the 28°C culture but was not reached until the 5th day in the culture oscillating between 26 and 28°C and the 6th day in the culture oscillating between 24 and 28°C. The cell growth phase in the culture oscillating between 24 and 28°C was about 2 days longer than that in the culture at a constant 28°C. The maximal cell densities of cultures oscillating between 22 and 28°C and those oscillating between 20 and 28°C were not as high as that of the 28°C culture (16.4 × 10⁵ and 14.3 × 10⁵ cells/ml versus 18.4 × 10⁵ cells/ml), whereas the viable cell density did not reduce at the 7th day in these two cultures. The effect of temperature oscillation on insect cell growth in suspension cultures is shown in Fig. 2. The culture with oscillation between 24 and 28°C gave the highest viable cell density (25.4 × 10⁵ cells/ml) among all the cultures, and its cell growth phase was 2 days longer than that at 28°C. These results indicated that temperature oscillation can prolong the cell growth phase. Oscillation between 24 and 28°C was optimal for promoting high cell viability without decreasing the maximal cell density.

FIG. 1.

Growth of uninfected insect cells at four temperature oscillations and one constant temperature in stationary culture (five replicates were done). Symbols: ○, 28°C; □, 26 to 28°C; ▵, 24 to 28°C; ▿, 22 to 28°C; ◊, 20 to 28°C. Error bars, standard deviations.

FIG. 2.

Growth of uninfected insect cells at four temperature oscillations and one constant temperature in suspension culture (five replicates were done). Symbols: ○, 28°C; □, 26 to 28°C; ▵, 24 to 28°C; ▿, 22 to 28°C; ◊, 20 to 28°C. Error bars, standard deviations.

Effect of temperature oscillation on infected insect cell growth.

The growth of infected insect cells in stationary cultures at different temperature oscillations is shown in Fig. 3. The viability of infected cells declined quickly at 28°C and slightly more slowly with oscillation between 26 and 28°C at 1 day postinfection, whereas the viability of other cultures did not change much in the first 3 days postinfection. The culture with oscillation between 24 and 28°C kept the highest level of cell viability at 3 days postinfection. This suggested that cultures with temperature oscillation can maintain high levels of cell viability, even postinfection. Results with suspension cultures were similar to those with stationary cultures (Fig. 4). Temperature oscillation was beneficial for maintaining infected cell viability. When the lower limit of temperature oscillation was below 24°C, the viable cell density could be maintained for 3 days.

FIG. 3.

Growth of infected insect cells at four temperature oscillations and one constant temperature in stationary culture (five replicates were done). Symbols: ○, 28°C; □, 26 to 28°C; ▵, 24 to 28°C; ▿, 22 to 28°C; ◊, 20 to 28°C. Error bars, standard deviations.

FIG. 4.

Growth of infected insect cells at four temperature oscillations and one constant temperature in suspension culture (five replicates were done). Symbols: ○, 28°C; □, 26 to 28°C; ▵, 24 to 28°C; ▿, 22 to 28°C; ◊, 20 to 28°C. Error bars, standard deviations.

Effect of temperature oscillation on cell viability and virus infectivity.

To investigate the effect of temperature oscillation on virus infection, cell viability and virus infectivity were examined in the 28°C culture, the culture with oscillation between 24 and 28°C, and the culture with oscillation between 20 and 28°C. Figures 5 and 6 show the cell viabilities and virus infectivities in stationary cultures and suspension cultures, respectively. There was much similarity between the stationary culture and the suspension culture, except the infection rate in the suspension culture was a little higher than that in the stationary culture. This can be explained by the fact that the movement of released virus particles from infected to noninfected cells in suspension culture is easier than that in stationary cultures (5). When the rate of virus infection was high, the cell viability dropped rapidly at 28°C. Compared with the culture at 28°C, the culture with oscillation between 24 and 28°C had a higher cell viability and a slightly lower rate of virus infection. In the culture with oscillation between 20 and 28°C, virus infectivity remained below 80% and cell viability was nearly 40% at the 4th day postinfection. More time was probably needed to attain a higher infectivity in this oscillation culture.

FIG. 5.

Cell viability (open symbols) and virus infectivity (solid symbols) at two temperature oscillations and one constant temperature in stationary culture (five replicates were done). Symbols: ○ and •, 28°C; □ and ▪, 24 to 28°C, ▵ and ▴, 20 to 28°C. Error bars, standard deviations.

FIG. 6.

Cell viability (open symbols) and virus infectivity (solid symbols) at two temperature oscillations and one constant temperature in suspension culture (five replicates were done). Symbols: ○ and •, 28°C; □ and ▪, 24 to 28°C, ▵ and ▴, 20 to 28°C. Error bars, standard deviations.

Effect of temperature oscillation on virus production.

Table 1 indicates the virus productions in both stationary and suspension cultures under different temperature conditions. Among all experiment conditions, the oscillation between 24 and 28°C was optimal for virus production in terms of the titer of ECV and the number of OB. The virus production in the culture with oscillation between 24 and 28°C was significantly higher than that of the 28°C culture by the t test for independent samples (Table 1). The titer of ECV was increased 18.4% on a volumetric basis and 31.0% on a cellular basis in the stationary culture. These increases were 23.0 and 44.3%, respectively, in the suspension culture. The number of OB was increased 16.7% on a volumetric basis and 30.0% on a cellular basis in the stationary culture, and these increases were 10.7 and 30.2%, respectively, in the suspension culture. The culture with oscillation between 26 and 28°C also produced more ECV and OB on a cellular basis than the culture at 28°C did. Compared with the 28°C culture, the virus production of the culture with oscillation between 22 and 28°C was no less on a cellular basis and less on a volumetric basis. The culture with oscillation between 20 and 28°C culture produced less virus than the 28°C culture at the 4th day postinfection. Since the virus infectivity was below 80% at that time, the virus production in the former culture might be increased if its time were prolonged.

TABLE 1.

Baculovirus production under different temperature conditions in stationary and suspension cultures^a

Culture	Temper- ature (°C)	No. of OB produced		ECV titer
		107/ml	Cell−1	107 PFU/ml	PFU/cell
Stationary	28	2.10 ± 0.061 C	13.4 ± 0.39 C	13.6 ± 0.20 C	87 ± 1.30 D
	26–28	2.21 ± 0.063 B	14.8 ± 0.43 B	14.8 ± 0.20 B	99 ± 1.30 B
	24–28	2.45 ± 0.083 A	17.4 ± 0.59 A	16.1 ± 0.26 A	114 ± 1.84 A
	22–28	1.72 ± 0.058 D	13.0 ± 0.44 C	12.1 ± 0.30 D	92 ± 2.20 C
	20–28	1.33 ± 0.056 E	11.0 ± 0.46 D	9.6 ± 0.26 E	79 ± 2.15 E
Suspension	28	2.60 ± 0.05 B	13.9 ± 0.28 C	14.8 ± 0.26 C	79 ± 1.40 D
	26–28	2.63 ± 0.053 B	14.9 ± 0.30 B	16.0 ± 0.20 B	90 ± 1.13 C
	24–28	2.88 ± 0.054 A	18.1 ± 0.34 A	18.2 ± 0.17 A	114 ± 1.09 A
	22–28	1.67 ± 0.041 C	13.5 ± 0.33 C	11.7 ± 0.17 D	94 ± 1.40 B
	20–28	1.38 ± 0.045 D	11.7 ± 0.38 D	9.6 ± 0.26 E	81 ± 2.24 D

^aValues are mean productions of OB (based on five replicates) and of ECV (based on three replicates) ± standard deviations. For values followed by the same letter, the means are not significantly different (P < 0.05), as determined by the t test for independent samples. 

DISCUSSION

Our results show that temperature oscillation can prolong the cell growth phase of uninfected and infected sf9 cells in stationary and suspension cultures. The optimal oscillation for promoting a long cell growth phase without decreasing the maximal cell density was between 24 and 28°C. At temperatures below 22°C, cells grew too slowly (8) and did not reach a cell density as high as that reached in the 28°C culture. An additional reason for the long phase of cell viability postinfection was the low infection rate due to the low temperature (8). As quick infection at high temperatures leads to quick cell lysis, implementing a suitable temperature oscillation was able to increase baculovirus production. There are two possible explanations for this: either infected insect cells survive longer at temperatures oscillating from low to high so that they can produce more virus or the slow infection that results from temperature oscillation might leave some viable cells to divide, causing secondary infection (6, 7) and thus increasing virus production.