To begin, place the trypsinized Sf1Ep cell culture and a 6-well plate on a working platform. Add the appropriate number of Sf1Ep cells and Sf1Ep medium to each well of a 6-well plate. Incubate the plates at 37 degrees Celsius with 5%carbon dioxide overnight.
Combine the components of TpCM-2 in a sterile container, adding dithiothreitol as a dry powder last. Gently mix and filter sterilize the medium using a 0.22 micrometer filter unit. Pre-equilibrate the medium in an anaerobic jar evacuate the chamber using house vacuum and refill with a 95%nitrogen and 5%carbon dioxide mixture three times.
Then refill with 1.5%oxygen, 5%carbon dioxide and balance nitrogen gas mixture. Quickly transfer the medium to a tri-gas incubator, set up at 34 degrees Celsius and incubate overnight. After overnight incubation, remove the medium from the Sf1Ep culture and rinse the cells with a small volume of the pre-equilibrated TpCM-2.
After removing the rinse, add an appropriate amount of TpCM-2 and equilibrate the plate in 1.5%oxygen, 5%carbon dioxide, and balance nitrogen atmosphere at 34 degrees Celsius for three to four hours. Remove the existing T.pallidum culture from the low oxygen incubator and examine the Sf1Ep cell layer in each well. After recording the cell density and appearance, pipette the medium from each well into a sterile 15 milliliter conical tube.
Rinse each well with 0.35 milliliters of prewarmed trypsin-EDTA and add the rinse to the 15 milliliter tube. Add another 0.35 milliliters of trypsin-EDTA to each well and incubate for five minutes at 37 degrees Celsius to remove Sf1Ep cells from the plate and T.pallidum from Sf1Ep cell. After detachment, combine the dissociated T.pallidum and Sf1Ep cells with the reserve medium collected in the 15 milliliter conical tube and record the total volume retrieved for calculation of the yield per culture.
Inoculate the previously prepared plate of Sf1Ep cells with a set volume of the harvested T.pallidum. After exchanging the atmosphere in the plate, incubate at 34 degrees Celsius within the brewer jar or in a tri-gas incubator. After inoculation, use a helper counting chamber to enumerate T.pallidum under dark-field microscopy.
Add 50%glycerol to a final concentration of 10%to the T.pallidum culture and disperse the glycerol through gentle pipetting or inversion. Distribute the preparation in one to two milliliters aliquots into screw capped freezer vials, and immediately place the vials in a 80 degrees Celsius freezer or a liquid nitrogen freezer. Prepare and pre equilibrate two 96-well plates with 1000 Sf1Ep cells and 200 microliters of TpCM-2 per well and incubate overnight in a tissue culture incubator.
The next day, replace Sf1Ep medium with TpCM-2 as described previously. After quantitation, dilute T.pallidum in TpCM-2 to produce two preparations with concentrations of 10 and 40 cells per milliliter. Inoculate 50 microliters per well of the 10 T.pallidum per milliliter preparation into one of the prepared 96 ell plates.
In the two control wells in each plate, inoculate the 2 x 10 to the power of three T.pallidum per milliliter dilution. Equilibrate the plates with the low oxygen gas mixture and incubate them in a brewer jar or tri-gas incubator at 34 degrees Celsius. On the seventh day, remove half of the medium and replace it with fresh equilibrated TpCM-2.
Once positive wells are identified by dark-field microscopy, trypsinize the positive wells and transfer the cell suspension to the previously prepared 24-well plates for further expansion. T.pallidum typically retain over 90%motility and multiply logarithmically with a doubling time of 33 to 45 hours for approximately seven days before entering the stationary phase. Over the course of one week, the spirochetes underwent approximately four to five doublings.
