In the current studies, we established a variety of proliferating cell lines from Ascalapha odorata egg tissue. After generating primary cultures, proliferating cells were identified as individual colonies at approximately 77 and 90 days. Cells from proliferating colonies were used for single cell cloning by limiting dilution at this relatively early stage (13 days after identifying three foci of proliferating cells in culture 2.0AA) in order to preserve the diversity of potentially useful cells. The 23 rapidly growing cell lines exhibited variations in size, shape, substrate adherence and growth rates. In a preliminary screen, cloned cells were examined based on growth characteristics, AcMNPV infectivity, and production of recombinant secreted alkaline phosphatase (SEAP) from a baculovirus vector. From these, one cell line (Ao38) was selected for more detailed analysis.
Ao38 cells grown in TNMFH are moderately-to-tightly adherent, spindle shaped, and are relatively large in comparison with the more commonly used Sf9 cells. Measurements of packed cell volumes indicated that Ao38 cells are larger than Sf9 cells, but smaller than High Five cells. Ao38 cells have a morphology similar to that of High Five cells (Fig. ). Our subsequent analysis showed that Ao38 cells had the following culture characteristics. Ao38 cells can be plated at low densities (approximately 5% confluency) without substantial effects on growth rate, and cells grow rapidly (doubling time of approximately 20.2 h) to relatively high densities of over 1.4 × 106 cells/ml in stationary cultures. As cells grow beyond confluency, some vertical aggregation of cells was observed. The observed clumping may result from a lower level of contact inhibition than observed in other cell lines, and that may contribute to the observation of higher cell densities in adherent and suspension cultures and continued recombinant protein production. Cell clumping was minimized by stepwise adaptation to suspension culture: removing cell aggregates at intervals.
Perhaps the most important and widespread applications of insect cell lines involve the production of secreted proteins that will be later purified from the growth medium. Therefore growth of cells and production of recombinant proteins in serum-free medium is an important characteristic. Ao38 cells were adapted to a commercial serum-free medium, Sf-900III, within a relatively short time period (12 days) and without laborious or stepwise adaptation. However, the Sf-900III-adapted cells did not grow successfully in three other commercial media that have been used for Sf9 and/or High Five cells. Ao38 cells also adapted readily to shaker cultures, using 5 steps to remove aggregates of cells during the first 15 days of growth in the suspension cultures. The procedures for adaptation to suspension cultures were similar to those we have previously used for High Five cells. Cell growth curves in suspension cultures and cell viability assays indicated that Ao38 cells grew optimally in shaker cultures at cell densities ranging from 0.2 × 106 to 2.5 × 106 cells per ml.
We also examined Ao38 cells in terms of viral infectivity, yields, and production of recombinant proteins. Comparisons with Sf9 and High Five cells revealed that Ao38 cells were similar to Sf9 cells in susceptibility to WT AcMNPV infection and both were less susceptible than High Five cells. In contrast to viral susceptibility studies, we found that yields of infectious virus were highest in Sf9 cells. WT AcMNPV yields from Ao38 cells were slightly less than observed from Sf9 cells, but higher than virus yields from High Five cells. Thus, in comparisons of viral susceptibility and virus yield in Sf9 and High Five cells, Ao38 cells compare favorably and are intermediate between these two well-established cell lines. The kinetics of β-galactosidase and SEAP protein production also suggest that progression of the infection cycle is similar in the three cell lines although future studies will examine this in more detail. More importantly though, recombinant protein yields from AcMNPV-infected Ao38 cells were higher those from Sf9 and High Five cells. While the kinetics of β-galactosidase production were generally similar among the three cell lines, peak protein levels were substantially higher from Ao38 cells. Although β-galactosidase production in High Five and Sf9 cells appeared to decrease after 4 days, we found that β-galactosidase continued to accumulate through 6 days post infection in Ao38 cells. A decrease in intracellular β-galactosidase activity in Sf9 and High Five cells after 4 or 5 days p.i. has been previously reported [6
]. Production of peak levels of the secreted protein, SEAP, was most rapid in High Five cells. However, peak SEAP levels in Ao38 cells were approximately 50% greater than peak SEAP levels in High Five cells (Fig. ). This may be explained by the observation that SEAP production in Ao38 cells appears to continue over a longer time period, permitting accumulation of higher quantities of the secreted protein. It was previously reported [15
] that on a culture volume basis, High Five cells produced at least fivefold more SEAP than Sf9 cells. The maximum concentration of SEAP reported for High Five cells was 9.5 U/ml. These results are comparable to SEAP data from the current study (Fig. ). We measured peak SEAP production in High Five cells at 15.5 U/ml whereas peak SEAP production in Ao38 cells was measured at 23.2 U/ml in serum-containing medium, and 20.9 U/ml in serum-free medium. Overall our analysis of protein production using two reporter proteins expressed from recombinant baculoviruses indicates that Ao38 cells are capable of producing higher levels of both intracellular and secreted proteins. The reason for higher level protein production by Ao38 cells is unclear although it may result from either more robust protein synthetic machinery, more efficient protein transport and processing, or some combination of these factors. Alternatively, our preliminary observations suggest that when compared with Sf9 and High Five cells, Ao38 cells appear to remain viable for a longer period after infection initiates, possibly resulting in a prolonged period for protein production. Future studies will address these and additional questions.
Insect cells are known to produce glycoproteins with paucimannnosidic or oligomannose N-glycan structures and this is believed to be due to either divergence of N-glycan processing pathways in subcellular organelles, and/or different regulation of the enzymes responsible for generating complex N-glycan structures [16
]. To examine and evaluate protein glycosylation in Ao38 cells, we performed LC-MS/MS analysis on the N-glycan of recombinant SEAP protein produced from an AcMNPV expression vector. Based on the consensus sequence for N-linked glycosylation, SEAP has three potential N-glycosylation sites at N136
, and prior studies suggested that only the N266
site was utilized for glycosylation [12
]. In the current study of Ao38 cells, we applied the precursor ion scans monitoring the specific marker ion (HexNAc+
at m/z 204) in LC-MS/MS analysis for selective identification of all glycan isoforms associated with each of peptides. We identified N-linked glycosylation of two tryptic peptides at position N139
which were eluted at 8.5 min and 20 min, respectively under our LC conditions (Additional file 1
). This analysis is complementary with previous approaches for released glycans from SEAP protein, allowing us to determine the glycoisoforms and the difference for each of the N-glycan sites. Sugar composition at both N-glycan sites was paucimannosidic or oligomannose, but more mannose residues were present in the N-glycan at N266
. Interestingly, fucosylation at N-acetylglucosamine linked to Asn selectively occurred for the N-glycan at N139
. It is possible that the selective fucosylation may have occurred due to the nature of the structure around N139
. The nature of the fucose linkage (alpha 1,6 or alpha 1,3) in SEAP from Ao38 cells is not known but insect cell lines have been reported to differ in whether or not their glycoproteins contain core alpha (1,3) fucose (reviewed in reference [17
]), and fucosylation may have significance in various applications. While recombinant glycoproteins with a core alpha (1,3) fucose may not be problematic as therapeutics [20
], they have resulted in false positives when used in diagnostic assays [21
]. It will be of interest in future studies to determine whether this specific linkage is found in glycans from Ao38 cells.
Glycans at the N139
residue were predominantly Man3F and Man2F in composition (Additional file 1
). In contrast, Man3 and Man6 are the dominant glycan isoforms found at the N266
residue. Nevertheless, the identification of the glycans in SEAP expressed in the Ao38 cell line reported in this work is similar to that identified previously from Sf9 and High Five cell lines [10
]. It should be noted that the relative quantitation of identified N-linked glycan isoforms should be treated as an estimate since the assumption that all sugar isoforms share a similar ionization efficiency may not be accurate.
Recently an adventitious virus was identified in High Five cell lines. The alphanodavirus, TNCL virus, was identified during production of hepatitis E virus-like particles in High Five cells infected with a recombinant baculovirus vector [13
]. Using RT-PCR and primers specific for five regions of the TNCL virus genome, we were unable detect the TNCL Virus in AcMNPV infected Ao38 cells, an uncloned parental cell line (AoP) from Ascalapha odorata
, or Sf9 cells (a negative control) (Fig. ). However, TNCL virus was detected in AcMNPV-infected High Five cells (passage 98) used as a positive control. Persistent viral infections have been observed occasionally in insect cell lines [22
] and nodaviruses are thought to occasionally infect wild marine invertebrates either latently or persistently [24
]. A betanodavirus was also reported to be latently infecting a cell line derived from the brain tissue of the barramundi fish, Lates calcarifer