2.2.1 Host Cells Used for Cell Culture
The cell culture technique used was one developed by AgVax Developments Ltd for the continuous passage of Toxoplasma gondii for ToxoVax production. Vero cells, originating from monkey kidney, were provided by AgResearch at Wallaceville, Upper Hutt and used as the primary cell line. Additionally, bovine endothelial cells were used as a cell line for cultures and were provided by the Institute of Veterinary, Animal and Biomedical Sciences (IVABS) at Massey University, Palmerston North.
2.2.2 Passage of Host Cells
A flask of host cells became 100% confluent once the cells had grown over the entire flask surface. Once cells become confluent they were passaged. Cell lines were passaged 1-2 times per week. Growth media was changes 2-3 times per week. Host cell lines were adherent cells and therefore required harvest from the flask surface, either mechanically using a rubber policeman or using enzymatic treatment with trypsin solution (Appendix 2.1 & Appendix 2.2).
At cell harvest, supernatant was tipped off and the cell layer was washed gently with two changes of PBS (Gibco) that was discarded. Cells were harvested by antibiotic-trypsin-versene (ATV) treatment for 5 min at 37°C, 1ml of ATV per 25 cm2
flask surface area or enough to lightly cover the cell surface area. Following the initial 5 min incubation at 37°C, flasks were tapped to encourage cells to detach from their surface. ATV flasks were incubated for a further 1-5 min if cells had not properly detached. Cells were washed from 25 cm2 flasks using 2 x 5 ml volumes of complete media (cMEM, made of 10% FBS in MEM plus antibiotics), which was retained. The ATV-cell suspension was centrifuged at 200 x g for 5 min. The supernatant was discarded and the cell suspension pipetted to break up cell clumps. Cells were washed again in cMEM before counting and dispensing aliquots into flasks with fresh growth media (cMEM).
Cells were counted using a haemocytometer following harvest. Cell lines were seeded at 50,000 cells per cm2 of flask surface. Cell monolayers, used to culture tachyzoites, were seeded at 30,000 cells per cm2 of flask surface. Cell seeding volume was calculated using the following formula: Volume = (required cell number (50,000 or 30,000) x flask surface area) / cell concentration.
2.2.3 Growth Media and Supplements Used for Culture
All cells were grown in Minimum Essential Medium (MEM) from Gibco (Invitrogen Corp NZ). Medium was stored at 4°C once opened. Growth medium was warmed to 37°C prior to addition to cultures. Cells were always washed with 2 changes of PBS prior to the addition of fresh media. Growth media for cell lines and monolayers prior to inoculation were supplemented with 10% FBS (Gibco) or HS (Gibco). Cells were maintained in either FBS or HS but the sera were not used interchangeably. The primary serum used was FBS.
Following inoculation of a monolayer with parasites, the media was supplemented with only 2% serum, whether FBS or HS. Both N. caninum and T. gondii tachyzoite cultures were maintained in 2% serum supplemented MEM.
All cultures, cell lines, monolayers and inoculated monolayers were maintained in the presence of antibiotic-antimycotic (50ug/ml penicillin, 50 ug/ml streptomycin and 50 ug/ml amphotericin) from Gibco (Invitrogen Corp NZ) and 50 ug/ml of gentamycin (Serva- research grade, provided by AgResearch, Wallaceville).
Growth media or complete media (cMEM) consists of MEM supplemented with 2 or 10% serum and antibiotic-antimycotics.
2.2.4 Growth Conditions for Cells and Parasites
2.2.5 Cytopathic Effect (CPE)
Cytopathic effect (CPE) could be identified by eye as a small clear dot in the cell monolayer. Parasitic movement around the area of CPE could be observed at 100x magnification. At 400x magnification parasites were readily distinguishable from surrounding debris and cells due to their characteristic shape and movement.
2.2.6 Passage of Toxoplasma gondii for Technique Development
Toxoplasma tachyzoites were initially used as a control parasite during the development of the cell culture techniques because a N. caninum strain was not available within New Zealand at that time.
Toxoplasma infected flasks were split when approximately 90% of the monolayer was infected with parasites. Parasites were inoculated onto new monolayers every 1-2 weeks or when CPE reached 75-90%. Media was changed 2-3 times per week. The procedure described for N. caninum passage was also used for T. gondii.
2.2.7 Passage of Neospora caninum Tachyzoites
Neospora caninum parasites were imported into New Zealand approximately six months following study commencement. Nc-Liverpool (isolated in England) was imported from Sydney, Australia. Dr John Ellis from the University of Technology, Sydney, kindly provided the Neospora parasites isolated by Prof. Trees at the University of Liverpool and the cooperation of both is acknowledged.
Parasites were primarily maintained in Vero cells supplemented with 2% FBS and antibiotics, which are the growing conditions used by Barber et al., (1993) following parasite isolation, with the exception of MEM being used instead of RPMI-1640. Parasites were also grown in a bovine endothelial host cell line which was maintained in the same manner as the Vero cell line.
Parasites were passaged onto a new monolayer every 1-2 weeks or when CPE reached 75-90%. Media was changed 2-3 times per week. Flasks were split using ATV as described for host cell lines. A split ratio of 1:3 to 1:5 was used to passage N. caninum (and T. gondii) infected cultures onto new monolayers. However, during culture trials parasites were counted and inoculated onto monolayers at known parasite densities.
2.2.8 FBS Evaluation
Foetal bovine serum (FBS) was tested for anti-N .caninum antibodies using a commercially available indirect fluorescent antibody test (IFAT) using a commercial test performed by AgriQuality, Palmerston North. All serum was heat inactivated at 56°C for 30 min to inactivate complement prior to testing. A minimum of 1 ml of serum was sent for each sample tested.
Several samples from the same bottle of FBS were also sent in some cases. Samples were submitted for serological testing on three separate occasions.
2.2.9 Serum Trial
Foetal bovine serum (FBS) and horse serum (HS) were used to study the growth rate of Vero cells and N. caninum parasites in order to evaluate whether anti-Neospora antibodies present in FBS affected N. caninum growth in culture. On day zero, 112 x 25 cm2 monolayers were prepared from pooled Vero cells that had previously been grown in FBS. The flasks were seeded at 30,000 cells/cm2, which totalled approximately 750,000 Vero cells per flask. On day 1, the 24- hour monolayers were divided into 4 groups. All monolayers were cultured in 10% serum MEM and maintained at this level throughout the trial. All monolayers were washed with PBS and cultured as follows: Group 1; Vero cells cultures in 10% FBS, Group 2; Vero cells cultured in 10% HS, Group 3; Vero cells inoculated with 2.5x106 N. caninum tachyzoites and cultured in 10% FBS, Group 4; Vero cells inoculated with 2.5x106 N. caninum tachyzoites and cultured in 10% HS. On days 4, 7, 10 and 13, three flasks from each group were rinsed twice with PBS, harvested using ATV, centrifuged at 200 x g for 10 minutes, resuspended into growth media and cell counts were performed for Vero cells and N. caninum tachyzoite. On the same days, all other remaining flasks were rinsed twice with PBS and the respective growth media were replaced. From day 7 onwards, the supernatant and wash media from all N. caninum infected cultures was also collected, centrifuged at 200 x g for 10 minutes, resuspended into growth media and either counted, for flasks that were harvested for analysis, or for all other flasks and supernatant captured tachyzoites were added back into the flask they were removed from for further culturing. Supernatant harvest was performed in order to maintain the correct parasite count for each flask throughout the trial.
2.2.10 Percoll Density Gradient Separation of Tachyzoites
A 30% Percoll-PBS solution was made and the pH tested using litmus paper. Nc-Liverpool tachyzoites were harvested from culture and observed under a microscope for signs of activity and viability. Tachyzoites were resuspended into the 30% Percoll-PBS solution and a sample of resuspended tachyzoites was observed under a microscope for signs of distress. The remaining parasites were centrifuged at 2,700 x g (3,400 rpm) in a bucket centrifuge for 10 minutes. The supernatant was removed with a pipette so as not to disturb the pellet and was examined microscopically for tachyzoite presence. The cell pellet was harvested, washed once and resuspended in growth media. A sample was taken from the resuspended solution and a microscope was used to identify tachyzoites. The remaining resuspended cell pellet was inoculated onto a 24-hour monolayer and incubated to validate parasite viability. The procedure used was based on that described by (McGuire et al., 1997a).
2.2.11 Cryopreservation of Tachyzoites
The cryopreservation procedure used for the N. caninum was based on the technique described by (Barber et al., 1995). Flasks were harvested for cryopreservation when many parasite clusters
were visible but little CPE could be seen, usually 2 days P.I. Cell layers were scraped from the flask surface using a rubber policeman (flasks were not treated with ATV). Cells were not disrupted by pipetting following harvest. The cell solution was centrifuged at 200 x g for 10 min. The sediment from each 25 cm2 flask was resuspended into 2 ml of chilled cryopreservation mixture (5% serum (Gibco), 10% DMSO (Sigma), 85% MEM (Gibco)) and aliquoted into 2 x 1 ml cryotubes. Cryotubes were placed into an isopropyl alcohol cryo 1°C freezing container for a minimum of 3 hours or overnight before transfer to a liquid nitrogen Dewar. Tubes were placed onto cryo-straws and secured with a plastic sheath before placing into a liquid nitrogen Dewar. Cultures were retrieved from cryopreservation by immersing the frozen vial in a 37°C water bath with mild agitation for 2-3 minutes (the lid of the vial was not immersed). The contents were removed from the tube, resuspended in PBS and centrifuged at 200 x g for 10 minutes. The cells were washed in PBS twice. Sedimented cells and parasites from each vial were resuspended in 2 mls per vial of 2% growth media and inoculated onto a 175 cm2 monolayer (1 x cryopreserved vial: 175 cm2 monolayer).
2.2.12 Cryopreservation Trial
A 25 cm2 flask of N. caninum infected Vero cells was harvested 4 days after inoculation with N caninum. Small spots of CPE could be seen in the monolayer using a microscope but CPE could not be seen clearly by eye. Cells were harvested from the flask surface using a rubber policeman and resuspended into 2% FBS-MEM. Half of the cell solution was removed and plated onto a 24- hour monolayer. The other half of the cell solution was processed as described above and cryopreserved for 2 days. Following two days of storage at -196°C, the cells were retrieved from cryopreservation as described above and inoculated onto a 24 hour monolayer. The monolayer inoculated with parasites that had not been cryopreserved was harvested 7 days after seeding. The parasites were enumerated using a haemocytometer. The monolayer inoculated with cryopreserved parasites was also harvested 7 days after seeding and the parasites counted.
The same procedure was repeated for flasks of N. caninum infected Vero cells harvested 9 days after inoculation.