Procedures for In Vitro Cultivation of Treponema pallidum, the Syphilis Spirochete

Much of our research is directed at gaining a better understanding of T.pallidum metabolism. Understanding its growth requirements will facilitate so many aspects of syphilis research. Understanding of the T.pallidum physiology, structure, host immunity, antimicrobial susceptibility, and perhaps, ultimately, could lead to new methods of syphilis control.

One of our biggest challenges is the presence of mammalian cells in the culture system. If we can determine what nutrients the cells are providing, we can remove them from the system and it would simplify it. That would allow us to have no contaminated mammalian-cell DNA or protein, which would make the experimental analyses easier.

For over 100 years after its initial isolation, T.pallidum was propagated through infection of rabbits. The in vitro culture system allows you to avoid the use of animals and is more cost effective. It also allows for techniques such as genetic engineering that would not be possible in the animal model.

The availability of an in vitro culture method has already greatly expanded the number of groups studying Treponema pallidum. It has also led to advances such as the first mutagenesis of the organism, as well as high-throughput screening of antibiotics for those useful in treating syphilis. In vitro culture has paved the way for the detailed analysis of T.pallidum growth requirements, gene function and immune targets.

These questions are difficult, if not impossible, to address effectively using just the rabbit model. We hope that these approaches will help improve the diagnosis, treatment, and prevention of syphilis. In addition to our efforts to improve the in vitro cultivation system, we will focus on genetic manipulation of T.pallidum through transposon mutagenesis.

We hope to do a large-scale functional screen to identify genes important in T.pallidum pathogenesis. To begin, place freshly-prepared prewarm Sf1Ep cell medium and thawed Sf1Ep cells in a cryo vial on a working platform. Add one milliliter of Sf1Ep cell medium to the cryo vial and mix gently.

Transfer the mixture to a sterile 15-milliliter conical centrifuge tube containing five milliliters of Sf1Ep cell medium and gently mix. Centrifuge the cell suspension at 100 g for seven minutes and discard the supernatant before resuspending the pellet in 15 milliliters of fresh Sf1Ep cell medium. Transfer the cell suspension to a T75 tissue culture flask and place it in a standard humidified tissue culture incubator for one week.

After incubation, aspirate the medium from the flask and rinse the cell layer with five milliliters of sterile PBS. After aspirating the PBS, add 2.5 milliliters of trypsin-EDTA to the flask and seal the cap. Incubate the flask at 37 degrees Celsius for five minutes.

Tap the flask gently to dislodge the cells and observe under an inverted microscope to confirm the dispersal of the cells. Add five milliliters of Sf1Ep growth medium and rock the flask to mix with the trypsin-EDTA. Transfer the cell suspension to a conical tube.

And quantitate the cells using a hemocytometer or automated cell counter. To freeze Sf1Ep cells, spin the cells at 100 g for seven minutes and resuspend the pellet in Sf1Ep medium supplemented with 10%DMSO. Distribute one milliliter of cell suspension to each cryo vial and freeze overnight at 70 to 80 degrees Celsius.

To begin, place the trypsinized Sf1Ep cell culture and a six-well plate on a working platform. Add the appropriate number of Sf1Ep cells and Sf1Ep medium to each well of a six-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 prewarm 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 cells. 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 Helber 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 1, 000 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-well plates.

In the two control wells in each plate, inoculate the two times 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.