Host Cell Density Research Articles

Rational scale‐up of a baculovirus‐insect cell batch process based on medium nutritional depth

We have developed a serum-free cell culture process utilizing a recombinant baculovirus (AcNPV) expression vector to infect Trichoplusia ni insect cells for the production of the human lysosomal enzyme, glucocerebrosidase. The enzyme, which is harvested as a secreted protein in this process, can serve as a replacement therapy for the genetic deficiency Gaucher disease. In the course of pilot scale-up of a batch glucocerebrosidase process from 25-mL working volume shaker flask units to 25-L working volume stirred bioreactor units, a semi-empirical model was developed for the rational determination of scaleable process parameters, including host cell density at infection, multiplicity of infection (MOI), and harvest time. A key assumption of the model is that maximum protein production is limited by the serum-free medium's nutritional capacity, which can, in turn, be determined from the growth of uninfected cells. For the host cell/medium combination used in this study, the nutritional limit was determined to be 1.3 × 107 to 1.7 × 107 viable-cell-days/mL. Based on this, the model predicts that optimal protein expression is consistent with a 4-day batch process where the host cell density at the time of infection is 1.5 × 106 to 2.0 × 106 cells/mL and the MOI is 0.09–0.3. These parameters were empirically confirmed to give the highest achievable batch product yield, first in shaker flasks and then at larger scales. The low MOI allows at least one population doubling to take place post viral addition, so that the effective infected cell density producing product generally exceeds 4 × 106 cells/mL. It was also interesting to note that this process consistently achieved the same level of maximum protein production at the 25-L bioreactor scale in 4 days compared to 5 days at the shaker flask scale. This may be attributable to better control of the culture environment in the bioreactor. Unlike some other lepidopteran insect cells, such as Sf-9, T. ni cells were found to produce significant levels of the inhibitory metabolites ammonia and lactate. Our results suggest that reduction and/or removal of inhibitory metabolites might be beneficial for infection of high-density cultures of these cells and might also facilitate application of more sophisticated culture strategies, including fed-batch. © 1996 John Wiley & Sons, Inc.

Effects of Host Cell Density on Cell Infection Level in Antheraea eucalypti (Lepidoptera: Saturniidae) Cell Cultures Persistently Infected with Nosema bombycis (Microsporida: Nosematidae)

ABSTRACT. Spores of Nosema bombycis Y9101, isolated from the beet armyworm, Spodoptera exigua, were primed with an alkaline solution and inoculated into Antheraea eucalypti cell cultures. Infected cells were subcultured every five days at three cell densities (2.5 × 103, 5.0 × 103, and 1.0 × 104 cells/cm2). A difference was observed in the spread of N. bombycis Y9101 infection between low‐density and higher‐density cultures of host cells. The host cell density did not affect the productivity of secondary infective forms of the parasite. The principal factor determining the rate of microsporidian infection in vitro was the number of host cells existing within the reach of extruded short‐coiled polar tubes from spores germinated intracellularly.

Kinetic characterization of baculovirus‐induced cell death in insect cell cultures

The death process of baculovirus-infected insect cells was divided into two phases: a constant viability (or delay) phase characterized by a delay time (t(d)) and a first-order death phase characterized by a half-life (t(1/2)). These two parameters were used in conjunction with the n-target theory to classify the kinetics of cell death under various conditions, including different multiplicity of infection (MOI), host cell lines, virus types, incubation volumes, cell density and extracellular L(+)-lactate and ammonium concentrations. Two groups of kinetic effects were found: one characterized by a constant number of hypothetical targets and the other by decreased numbers of hypothetical targets. The first group includes effects such as MOI, virus types, and host cell lines. The second includes the effects of environmental perturbations, such as incubation volume, cell density, and extracellular concentrations of L(+)-lactate and ammonium. Although the underlying mechanisms of these effects are as yet unknown, the death kinetics of infected cells significantly affects the recombinant protein production. In general, foreign protein production does not correlate with the cell life after infection.

High‐level recombinant protein production in bioreactors using the baculovirus–insect cell expression system

In order to develop an efficient process for large-scale production of recombinant protein, various factors were studied which affect the productivity of Sf-9 (Spodoptera frugiperda) insect cells when using the baculovirus expression system. It was shown that upon infection with the Bac-BRV6L recombinant baculovirus, the level per cell of VP6 (a bovine rotavirus nucleocapsid protein) would drop 10-fold when host cell density at the time of infection increased from 2 x 10(6) to 3 x 10(6) cells/mL. The decrease was found to be totally reversible by culture medium renewal after infection, even when cells were infected at the stationary phase. Recombinant protein production was 4-6 times higher using TNMFH medium supplemented with 10% fetal bovine serum (FBS) than in IPL/41 serum-free medium. Fine-tuning of infection parameters in a 4-L surface-aerated bioreactor resulted in the production of typically 350 mg/L of VP6 protein, representing more than 25% of total cell proteins.

Selection for bacteriophage latent period length by bacterial density: A theoretical examination

In bacteriophage (phage), rapid and efficient intracellular progeny production is of obvious benefit. A short latent period is not. All else being equal, a longer latent period utilizes host cell resources more completely. Using established parameters of phage growth, a simulation of three successive phage lysis cycles is presented. I have found that high, but not low, host cell densities can select for short phage latent periods. This results from phage with short latent periods more rapidly establishing multiple parallel infections at high host cell concentrations, whereas phage with long latent periods are restricted to growth within a single cell over the same period. This implies that phage with short latent periods habitually grow in environments that are rich in host cells.

Effect of methylamine and monodansylcadaverine on the susceptibility of McCoy cells to Chlamydia trachomatis infection.

We used inhibitors of receptor-mediated endocytosis to study the mechanisms of infectivity, especially the uptake mechanism, of Chlamydia trachomatis for cultured cells. The effect of methylamine and monodansylcadaverine on the different stages of the chlamydial growth cycle in McCoy cells was examined. There was a dose-related decrease in the number of chlamydial inclusions in the presence of these agents. Monodansylcadaverine also decreased the chlamydia-dependent uptake of radioactive amino acids. The agents did not affect the attachment of chlamydiae to the cells, but they increased the protease-removable fraction of cell-bound chlamydiae. The amines reduced the number of inclusions when added at different times during the first 24 h of infection. However, this effect was influenced by host cell density, so that the effect of the amines at the early infectious phase was nullified in confluent monolayers, whereas, during later phases, the effect was comparatively independent of host cell density. This indicates that the amines have different modes of action at different infectious stages. The effect of the amines was reversible, and they had no effect on the infectivity of pretreated chlamydial elementary bodies. These experiments suggest that methylamine and monodansylcadaverine inhibit both the internalization of chlamydiae into McCoy cells and their intracellular development. These results are consistent with the hypothesis that chlamydiae utilize a constitutive cellular process, such as receptor-mediated endocytosis, to enter cells.

Inhibition of onset of overt multiplication of Chlamydia psittaci in persistently infected mouse fibroblasts (L cells).

When monolayers of mouse fibroblasts (L cells) persistently infected with Chlamydia psittaci (strain 6BC) were dispersed in medium 199 and plated out in new flasks, the monolayers that grew out consisted almost exclusively of inclusion-free host cells that retained full resistance to superinfection with C. psittaci (covert infection). After a delay that was inversely proportional to the initial density of the newly transferred L cell population, the percentage of host cells containing visible chlamydial inclusions increased rapidly (overt infection), and most of the L cells were destroyed by extensive chlamydial multiplication (wipeout), leaving only a few survivors to start new persistently infected monolayers. When persistently infected L cell populations grown in medium 199 were transferred to Eagle minimal essential medium, the onset of overt multiplication was strongly suppressed although covert multiplication of C. psittaci continued unabated, as shown by host cell retention of resistance to superinfection and the prompt resumption of overt multiplication after transfer back into medium 199. The difference(s) between the two media responsible for the different expression of the persistently infected state was not determined. A single dose of 100 U of penicillin G per ml of medium 199 given at the time persistently infected monolayers were divided almost completely suppressed the appearance of visible signs of chlamydial infection for several weeks, although resistance to superinfection was retained at all times. The same amount of penicillin given 7 days after replating did not prevent the occurrence of the first expected wipeout, but there was a long period of inclusion-free L cell growth between the first wipeout and the second. It was concluded that covert multiplication of C. psittaci in persistently infected L cells may continue indefinitely without the appearance of visible signs of infection. The transition between covert and overt chlamydial multiplication appears to be a penicillin-sensitive, multistep process that is regulated, at least in part, by the host cell density and the composition of the growth medium.

Persistent infection of mouse fibroblasts (McCoy cells) with a trachoma strain of Chlamydia trachomatis.

An in vitro model of persistent infection of mouse fibroblasts (McCoy cells) with a trachoma strain (G17) of Chlamydia trachomatis has been developed. Persistently infected cultures were established by infecting McCoy cells with high multiplicities of chlamydiae. After the first cycle of chlamydial replication, the host cells multiplied more rapidly than the parasites, so that the fraction of inclusion-bearing cells declined to less than 1%. However, after 100 days, the proportion of inclusion-bearing cells rose dramatically, and the cultures alternated between periods of massive host cell destruction by chlamydiae and periods of host cell proliferation. This cycle continued indefinitely as host cell and parasite densities fluctuated periodically. The chlamydiae in the cycling populations were reidentified as the original serotype. No changes in either host cell susceptibility or chlamydial invasiveness were observed in hosts and parasites recovered from persistently infected populations. All evidence suggests that the parasite maintained itself in McCoy cell populations by cell-to-cell transfer and that an equilibrium between host and parasite multiplication was achieved when the persistently infected cultures fluctuated between periods of host cell destruction and proliferation.