This work was supported by grant R01 AI141958 from the United States National Institutes of Health/NIAID. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Scalable In Vitro Cultivation of T. pallidum Using Mammalian Cell Co-Culture

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+TprK+profiling+of+in+vivo+and+in+vitro-propagated+Treponema+pallidum+subsp.+pallidum+reveals+accumulation+of+antigenic+variants+in+absence+of+immune+pressure.">Longitudinal TprK profiling of in vivo and in vitro-propagated Treponema pallidum subsp. pallidum reveals accumulation of antigenic variants in absence of immune pressure.</a> PLoS Negl Trop Dis. 15 (9), e0009753 (2021).</li><li>De Lay, B. D., Cameron, T. A., De Lay, N. R., Norris, S. J., Edmondson, D. 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The authors have no conflicts of interest to disclose.

The Sf1Ep-TpCM-2 system is the first available procedure that promotes the continuous in vitro culture of T. pallidum. The system is complex due to the extreme growth requirements of this organism: 1) complex nutritional needs because of limited biosynthetic capabilities; 2) a poorly understood requirement for small quantities of oxygen, despite high sensitivity to reactive oxygen species9,10,16,36; and 3) the current need for the presence of Sf1Ep cells. While it is tempting to &#39;cut corners&#39; on the procedure, it is recommended that the steps be followed carefully until a successful long-term culture is achieved prior to trying modifications. As additional information about the metabolic requirements of T. pallidum accumulates, it may be possible to develop axenic conditions that do not require the presence of Sf1Ep cells. However, the in vitro growth rate will likely remain slow (with a minimum doubling time of 33 h to 46 h, depending on the strain)17,18, given that T. pallidum multiplies at an estimated doubling time of 30 h to 33 h even during mammalian infection37,38. As with any bacterial culture, it is recommended that low passage stocks be maintained and that experiments be carried out with T. pallidum cultures that are less than 10 passages from these stocks to avoid &#39;genetic drift&#39; and associated phenotypic changes due to mutations.
Sf1Ep cells apparently provide essential nutrients or enzymatic activities to the treponemes. However, they also consume nutrients (such as glucose and oxygen) and may produce toxic conditions such as low pH9,16,39. Therefore, there is a balancing act between having sufficient Sf1Ep cells to support T. pallidum multiplication and preventing mammalian cell overgrowth and toxicity. High passage numbers of Sf1Ep cells tend to be faster growing and at times, lose the ability to support T. pallidum multiplication. As such, Sf1Ep passage number should be monitored, and cell stocks should be replaced with low-passage frozen preparations periodically. The presence of Sf1Ep cells also complicates the study of T. pallidum properties such as DNA, RNA, and protein content, and enzyme activities. Removal of the rabbit cells is possible to some extent using repeated low-speed centrifugation (100 x g for 5 min) or more effectively using Percoll or Hypaque gradients40,41. However, the gradient centrifugation methods are generally only effective with high numbers of T. pallidum. Alternative methods for propagating T. pallidum are limited to infection of laboratory animals such as rabbits13,14. This approach has ethical considerations and has become increasingly expensive; however, the rabbit model is very useful for studying T. pallidum pathogenesis and host immune responses. In addition, there are likely some differences in gene expression, growth or behavior of T. pallidum during in vitro culture and rabbit infection27.
At the time of this report, the Sf1Ep-TpCM-2 system has been established in at least 6 research groups in the United States and Europe and has resulted in 16 publications with topics ranging from T. pallidum basic biology and genetics to antimicrobial susceptibility. The value of in vitro culture in studying this enigmatic pathogen will likely increase with expanded usage and future improvements.

Treponema pallidum is a species of spiral-shaped bacteria (called spirochetes) that causes syphilis and related infections in humans and other primates. Syphilis is a serious disease with long-term effects on infected individuals, and it is estimated that over 8 million new cases of syphilis occur worldwide each year1. T. pallidum has been subdivided into three subspecies based on the diseases they cause in humans as well as minor genetic differences: subspecies pallidum (which causes the sexually transmitted disease syphilis), subspecies pertenue (yaws), and subspecies endemicum (causing bejel or endemic syphilis)2,3. T. pallidum subsp. pertenue also causes infections in baboons, chimpanzees, and other primates. A closely related organism called Treponema paraluiscuniculi (also called Treponema paraluisleporidarum) causes an infection in rabbits and hares4,5. All of these bacteria are very closely related, with greater than 98% DNA sequence identity at the genome level6,7,8. They each have a single, small circular chromosome about 1.14 million base pairs (Mb) in size. The members of this T. pallidum group are found only in association with their mammalian hosts; as such, they are obligate pathogens that are dependent on their host species for survival and growth9,10.
Attempts to culture T. pallidum in vitro began shortly after its identification by Schaudinn and Hoffman in 190511,12. However, these efforts failed to lead to consistent, reproducible growth of the organism. As a result, T. pallidum research studies required propagation of the organism through the experimental infection of laboratory animals, most commonly the rabbit13,14. In 1981, Fieldsteel et al.15 introduced a tissue culture system that promoted the multiplication of T. pallidum strains for a period of up to 2 weeks. This system involved co-culture of T. pallidum with Sf1Ep cottontail rabbit epithelial cells in a modified tissue culture medium (T. pallidum Culture Medium 1, TpCM-1) based on Eagle&#39;s Minimum Essential Medium (MEM) and 20% fetal bovine serum (FBS). Other culture conditions required were incubation at 34 &#176;C in an atmosphere containing 1.5% O2 and 5% CO29,16. In this system, T. pallidum attaches to the Sf1Ep cells and multiplies when in close association with the mammalian cell surface. Despite many subculture attempts and other modifications, the Fieldsteel et al. system failed to promote continuous in vitro growth.
In 2018, our laboratory reported that the use of a modified medium called TpCM-2 (in which Eagle&#39;s MEM was replaced with a more complex tissue culture medium, CMRL 1066) provided T. pallidum the required nutrients to permit consistent long-term culture17. To date, this modification has led to a consistent, continuous culture of at least 5 strains of T. pallidum subsp. pallidum (Nichols, SS14, Mexico A, UW231B, and UW249B) and one strain of T. pallidum subsp. endemicum (Bosnia A)18,19. As an example, the Nichols strain has now been cultured continuously in vitro for over 6 years. Thus far, attempts to culture yaws isolates (T. pallidum subsp. pertenue) or T. paraluiscuniculiin vitro have been unsuccessful18. The TpCM-2 system still requires the presence of Sf1Ep cells, low oxygen concentrations, and incubation at 34 &#176;C, making the system more complex than most bacterial culture techniques. However, this modified T. pallidum culture system has been useful for further defining the bacterium&#39;s growth requirements18, determining minimal inhibitory concentrations (MICs) of antimicrobial compounds and peptides20,21,22,23,24,25, propagating new strains from patient tissue aspirates26, isolating clonal populations of the organism27, characterizing the tprK antigenic variation system27,28, examining gene expression29, and performing mutational analysis30,31,32.
Here, we describe the current methods for cultivating T. pallidum in vitro. We hope this information will help facilitate the more widespread application of this in vitro culture technique to the improved diagnosis, treatment, and prevention of syphilis and related treponemal infections.

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		<tr>
			<td>0.5 M EDTA, pH 8.0</td>
			<td>Sigma&#160;</td>
			<td>E8008</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Earle&#8217;s Balanced Salts, w/o Mg2+, Ca2+</td>
			<td>Gibco</td>
			<td>14155063</td>
			<td></td>
		</tr>
		<tr>
			<td>15 and 50 mL conical sterile disposable centrifuge tubes&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL cryogenic vials&#160;</td>
			<td>Corning</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plates for T. pallidum cultivation&#160;</td>
			<td>Falcon</td>
			<td>353046</td>
			<td>The plates must have low evaporation lids.</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 tissue culture flasks with vented caps</td>
			<td>Corning</td>
			<td>43061U</td>
			<td></td>
		</tr>
		<tr>
			<td>93.5% nitrogen, 5% CO2, and 1.5% oxygen for pre-equilibrating medium and cultures</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>95% nitrogen and 5% CO2 for pre-equilibrating medium and cultures</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well low evaporation clear, flat-bottom tissue culture-treated microplates</td>
			<td>Corning Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable multi-channel pipette with 200 ul capacity&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Optional, but very helpful for cloning</td>
		</tr>
		<tr>
			<td>Cell culture grade water</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td></td>
		</tr>
		<tr>
			<td>CMRL 1066 without L-Glutamine or Phenol Red</td>
			<td>US Biological</td>
			<td>C5900-03A</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 for tri-gas and tissue culture incubators&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic liquid nitrogen cell culture storage tank</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable filter units, 0.2 &#181;m , &#62; 100 mL capacity</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable pipets: 25 mL, 10 mL, 5 mL, aspirating</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Mannitol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M1902</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO (sterile cell culture grade )</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle&#8217;s MEM</td>
			<td>Sigma-Aldrich</td>
			<td>M4655</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum, heat inactivated&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td>We highly recommend this product. Must pre-screen for T. pallidum culture compatibility if using a different brand or catalog number.</td>
		</tr>
		<tr>
			<td>Freezer with capability of maintaining -70 &#176;C or -80 &#176;C</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For storage of T. pallidum; liquid nitrogen storage may be used instead</td>
		</tr>
		<tr>
			<td>Freezing medium (Sf1Ep medium + 10% [v/v] DMSO)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas cylinders with appropriate fittings</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>GasPak 150 vented anaerobic jar (Brewer Jar)</td>
			<td>Fisher Scientific</td>
			<td>11-816</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Helber counting chambers with Thoma rulings</td>
			<td>Hawksley Medical and Laboratory Equipment</td>
			<td></td>
			<td>For quantitating T. pallidum</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>&#160;For Sf1Ep cell quantitation</td>
		</tr>
		<tr>
			<td>Incubator tank switch</td>
			<td>NuAire&#160;</td>
			<td>NU-1550 TankGuard Automatic CO2 Incubator Tank Switch</td>
			<td>Optional, but very helpful in maintaining appropriate O2 conditions.</td>
		</tr>
		<tr>
			<td>Inverted microscope with phase contrast optics</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For viewing Sf1Ep cell cultures</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>H6034</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope with darkfield condensor</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>The microscope should have a 40x objective and 15x eyepieces.</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M3183</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel adapter for aspirator&#160;</td>
			<td>Integra</td>
			<td>155520</td>
			<td>Optional, but useful for cloning</td>
		</tr>
		<tr>
			<td>NaHCO3 (7.5%)</td>
			<td>Sigma-Aldrich</td>
			<td>S8761</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen for tri-gas incubator</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin</td>
			<td>Sigma-Aldrich</td>
			<td>R7017</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf1Ep (NBL-11) cells</td>
			<td>American Type Culture Collection</td>
			<td>CCL-68</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile PBS (without calcium chloride and magnesium chloride)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile reagent reservoirs, 50 or 100 mL size</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>T. pallidum sample, frozen or fresh&#160;</td>
			<td></td>
			<td></td>
			<td>from a rabbit infection or in vitro culture</td>
		</tr>
		<tr>
			<td>Tissue culture incubator maintained at 37 &#176;C, 5% CO2</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-gas tissue culture incubator maintained at 34 &#176;C, 5% CO2, 1.5% O2</td>
			<td>Thermofisher</td>
			<td>Heracell&#8482; VIOS 160i Tri-Gas CO2 Incubator</td>
			<td>Optional; anaerobic jars may be used instead (see Ref. 17)</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum source (e.g. house vacuum), vacuum tubing, vacuum gauge, and connectors</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, suitable for cell culture, filter-sterilized, purified</td>
			<td>Sigma-Aldrich</td>
			<td>W3500</td>
			<td>Recommended for medium preparation; decreases culture variability</td>
		</tr>
	</tbody>
</table>

procedures for in vitro cultivation of treponema pallidum, the syphilis spirochete

For over a century, Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, could only be propagated by inoculation and harvest of the organisms from rabbit testes. In 2018, we described a method to continuously cultivate T. pallidumin vitro. This system utilizes co-culture with rabbit epithelial cells (Sf1Ep cells) in a serum-containing tissue culture medium called TpCM-2. The T. pallidum doubling time in culture is similar to that estimated to occur during natural infection (about 33-45 h). The organism can be cultured continuously with a standard passage time of 1 week in a low oxygen (1.5%) environment at 34 &#176;C. This article contains the protocols for culturing T. pallidum, methods for growing and maintaining the required tissue culture cells, and the technique for generating isogenic strains by limiting dilution. The ability to grow T. pallidum in vitro provides new experimental avenues to study and understand this enigmatic organism.

Author Spotlight: Advancing Syphilis Research &#8212; Innovations in Treponema pallidum Cultivation and Genetic Engineering

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 analysis 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.

Using the described conditions, T. pallidum typically retains&#62;90% motility and multiplies logarithmically with a doubling time of 33 h to 45 h for approximately 7 days before entering the stationary phase (Figure 3). Over the course of 1 week, the spirochetes undergo approximately 4-5 doublings (Figure 4). ). In addition, different strains of T. pallidum may grow at different rates. Strains of the SS14 group of T. pallidum tend to have slower doubling times than those of the Nichols group17.
Feeding of cultures may extend culture time by several days but the Sf1Ep cell layer often fails after a week of culture. Furthermore, the treponemes reach an upper limit of organisms of about 5 x 107/mL. Cultures transferred at 7 day intervals typically continue logarithmic multiplication with little or no lag phase. Organisms in the stationary phase often become difficult to pass.
Most T. pallidum are attached to the Sf1Ep cells. However, enough T. pallidum remains in the supernatant that medium samples can be removed periodically to check for viability and multiplication. If careful quantitation is required, the volume of the removed medium should be measured, the number of T. pallidum quantitated, and the total number of organisms removed added to the final counts at harvest.
In previous studies, the cloning efficiency (number of cultures positive per organism inoculated) were 12.5% for 2 T. pallidum inoculated per well and 6.7% for 0.5 T. pallidum inoculated per well27. Thus, it is likely that any positive well represents the outgrowth of a single organism at either of these inocula. However, the clonality of the resulting populations must be verified by examining the culture for homogeneity at sites that are heterogeneous in the parent culture. The most definitive way to demonstrate that the culture is isogenic is through whole genome sequencing27.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66880/66880fig01.jpg" /> 
Figure 1: Flow chart of the T. pallidumin vitro cultivation procedure. This figure has been reprinted with permission from Edmondson and Norris (2021)19. <a href="https://www.jove.com/files/ftp_upload/66880/66880fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66880/66880fig02.jpg" /> 
Figure 2: Diagram of the system to equilibrate T. pallidum culture reagents to a low-oxygen environment. A Brewer jar vent is connected via a T-joint to a vacuum source (such as a house vacuum) and to gas cylinders containing custom gas mixtures (5% CO2, balance nitrogen and 1.5% O2, 5% CO2, balance nitrogen). An in-line vacuum gauge measures the vacuum drawn in the jar. The vacuum is drawn in the jar to approximately -58 kPa. The evacuated jar is then slowly re-filled with the gas mixtures. The Brewer jar is refilled three times with 5% CO2, balance nitrogen before a final evacuation, and refilled with 95% N2, 5% CO2, 1.5% O2. Cultures or media are then removed from the jar and quickly transferred to the low-oxygen incubator. Alternately, the tubing between the Brewer jar and the first T-joint can be clamped tightly, the tubing disconnected from the T-joint, and the entire Brewer jar can be transferred to a 34 &#176;C incubator. This figure has been reprinted with permission from Edmondson and Norris (2021)19. <a href="https://www.jove.com/files/ftp_upload/66880/66880fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66880/66880fig03.jpg" /> 
Figure 3: Growth curves of T. pallidum cultured with Sf1Ep cells with TpCM-2 medium. Parallel triplicate cultures were seeded with T. pallidum. Replicates were harvested at each time point; the results represent the mean + SEM for these cultures. (A) The changes in T. pallidum per culture and (B) percent motility are shown. This figure has been adapted with permission from Edmondson et al.18. <a href="https://www.jove.com/files/ftp_upload/66880/66880fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66880/66880fig04.jpg" /> 
Figure 4: Example of passage of in vitro culture of T. pallidum, Nichols strain. Parallel triplicate cultures were seeded with T. pallidum and passed weekly using a 1:20 dilution. The sawtooth plot shows the numbers of T. pallidum per culture and the number transferred to new cultures at each time point. Results represent the mean &#177; SEM for three biological replicates. <a href="https://www.jove.com/files/ftp_upload/66880/66880fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Medium volume and seeding ratios for culture vessels. <a href="https://www.jove.com/files/ftp_upload/66880/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Media for T. pallidum cultivation. All media should be filter sterilized after preparation. Sf1Ep medium may be stored at 4 &#176;C for up to two months. TpCM-2 is typically made one day prior to use. The dissociation medium should be aliquoted and frozen. <a href="https://www.jove.com/files/ftp_upload/66880/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

This protocol describes in vitro cultivation of the syphilis pathogen Treponema pallidum subsp. pallidum in co-culture with mammalian cells. The method is scalable; it can be used to produce large quantities of T. pallidum and to generate clonal cultures.

For over a century, Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, could only be propagated by inoculation and ...

procedures-for-vitro-cultivation-treponema-pallidum-syphilis

Research

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JoVE Journal

Immunology and Infection

Establishing and Maintaining Sf1Ep Cell Stocks for Treponema pallidum Cultivation

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 minus 70 to minus 80 degrees Celsius.

In Vitro Cultivation of Treponema pallidum in Co-Culture with Sf1Ep Cells

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.

566 Views.  University of Texas Health Science Center at Houston - McGovern Medical School.  This protocol describes in vitro cultivation of the syphilis pathogen Treponema pallidum subsp. pallidum in co-culture with mammalian cells. The method is scalable; it can be used to produce large quantities of T. pallidum and to generate clonal cultures.

Scalable In Vitro Cultivation of T. pallidum Using Mammalian Cell Co-Culture

Summary