Standardization of an in vitro Drug Screening Assay by Use of Cryopreserved and Characterized Pneumocystis carinii Populations
In our laboratory, candidate anti-Pneumocystis carinii compounds are screened by a cell-free in vitro ATP-driven bioluminescent assay. This assay is based on the measurement of light generated by the enzyme substrate system of luciferase-luciferin in the presence of ATP. There is a linear relationship between the amount of ATP present, and the photons of light emitted. Historically, P. carinii isolated directly from the lungs of animal models have been used in this and other screening assays [4,5,6]. As a consequence, characterization of the population's karyotype, surface glycoproteins, microbial or host cell contamination status, or other variables of interest could not be assessed prior to use.
To better standardize the ATP assay, the feasibility of using cryopreserved organisms for screening assays was evaluated in the present study. ATP content and responses to standard anti-P. carinii compounds were compared between freshly isolated and cryopreserved organism populations. The results showed that the cryopreserved populations maintained ATP content over time and had increased sensitivity to anti-P. carinii compounds in a dose responsive manner, but not to control compounds such as ampicillin. Although the organisms were more sensitive to the compounds, they were comparable in their ability to select potentially efficacious compounds. Use of cyropreserved P. carinii allows characterization of the organism population prior to use in an entire series of experiments, which aids in standardization of the assay as well as decreases the investigator's reliance on the availability of infected animals.
MATERIALS AND METHODS
P. carinii organisms were obtained from Long Evans rats housed under standard conditions without barrier at the VAMC, Cincinnati Veterinary Medical Unit.Six-week-old animals were immunosuppressed using methylprednisolone (4 mg/ s.c. weekly, 8–10 weeks). This rat colony harbors single infections of P. carinii form 1 as well as co-infections of P. carinii form 1 and Pneumocyslis ratti [1]. Only P.carinii populations free of bacterial and fungal contaminants were used.
Freshly isolated organisms
P. carinii organisms were harvested from rat lungs using a modification of a previous protocol [2], Organisms were released from the tissue by mincing in RPMI-1640 medium, followed by homogenization for 10 minutes in a Stomacher Lab Blender (Tekmar, Inc. Cincinnati, OH). The homogenate was then sieved through sterile gauze and centrifuged at 2,000xg for 10 minutes, 4°C. Red blood cells were lysed with 0.85% aqueous ammonium chloride [pH 6.8] for 15 minutes at 37°C. Organisms were washed with RPMI-1640 as above and centrifuged at 18xg for 10 minutes at 4°C to reduce host cell contamination. The supernatant containing the P. carinii was removed and centrifuged at 2,000 ×g to collect the organisms. Organisms prepared for immediate use were treated with 10μg/ml DNAase l(Roche Diagnostics, Indianapolis, IN) for 5 minutes at 37°C, and washed twice in RPMI-1640. Pneumocystis to be assayed after cryopreservation was not treated with DNAase. Nuclei were enumerated by microscopic analysis of HEMA-3 (Curtis Matheson, Swedesboro, NJ) stained slides, using 10mm restricted diameter hydrophobic slides (Erie Scientific, Portsmouth, NH).
Cryopreserved organisms
Organisms to be cryopreserved were processed to remove host cells as described above and enumerated. The organisms were resuspended in RPMI-1640 containing 20% fetal calf serum to which an equal volume of RPMI-1640 with 15% dimethylsulfoxide was added [9]. Cryovials containing 1 ml of volume were stored at -20°C for 2 hours, then transferred to liquid nitrogen storage. Frozen stocks to be used for assay were rapidly thawed in a 37°C waterbath and washed in RPMI-1640 to remove DMSO. Organisms were then resuspended in assay medium.
Assay preparation
P. carinii organisms were suspended in an RPMI-1640-based medium supplemented with 20% calf serum, IX MEM vitamins, non-essential amino acids, L-glutamine, 100 IU penicillin and 100 μg/ml streptomycin. Concentration of nuclei was adjusted to 2 × 108/ml, and added to 48-well tissue culture plates.
Media with controls and 2× concentration of test compounds were added to triplicate organism populations, resulting in a final organism density of 1 x 108/ml and IX concentration of test compounds. At 24 hour intervals, wells were agitated, and 2% of the volume removed and extracted with 3.5% trichloroacetic acid to release the ATP. Extracts were stored at -20°C. To determine the ATP content of the samples, 10 μi of the extract was added to 750 μ1 of 0.1 M Tris, 2 mM EDTA [pH 7.75]. Luciferin-luciferase ATP monitoring reagent (Thermo Lab Systems) was automatically injected into the buffered sample and relative light units (RLU) recorded by an Autolumat LB 953 luminometer. Cytotoxicity was determined as percent reduction in test compound RLU/ampicillin control RLU. At each timepoint, organisms were fed by replacing 50% of the well volume with fresh media, with and without test compound.
RESULTS AND DISCUSSION
Three methods were evaluated to determine the optimal method of preparing the cryopreserved organisms after thawing: washing with RPMI; filtration through 10 μ Mitex filters to reduce debris; or slow speed centrifugation to remove debris. Organism viability was monitored throughout a 3-day period by the ATP method. All three methods produced organism populations that were viable and increased in ATP content over the assay period. Low speed centrifugation produced the highest initial ATP content which also increased 3-fold over time. While the viability of washed and filtered organisms was acceptable, low speed centrifugation was chosen as the optimal procedure for preparation of cryopreserved organisms after thawing.
The ATP levels of P. carinii after storage in liquid nitrogen (26 months) remained high throughout the evaluation period of 7 days (Table 1). ATP pools from untreated organisms doubled by day 1 and maintained this level until day 6. Organism numbers, as determined by enumeration of nuclei, also doubled by 24 hours, and slowly decreased after day 3. This kinetic profile has been previously observed in other studies [7]. The ATP reduction in the pentamidine-treated groups was dramatic, while nuclei counts did not begin to reflect the cytotoxic effect until day 4. This suggests that intact, but metabolically inactive nuclei can persist in the culture medium for several days, and produce misleading results in enumeration if enumeration is the sole criteria for assessment of activity. The reduction in ATP in organisms treated with pentamidine was similar to that of organisms isolated according to previous methods [2].
| Media | Pentamidine (1 μg/ml) | ||||
|---|---|---|---|---|---|
| # Nuclei | RLU | # Nuclei | RLU | Δ ATP (%)a | |
| Day 1 | 9.36 × 107 | 79,353 | 7.45 × 107 | 22,042 | 73.5 |
| Day 2 | 9.43 × 107 | 85,508 | 6.85 × 107 | 3,205 | 97.8 |
| Day 3 | 9.93 × 107 | 79,336 | 5.70 × 107 | 1,643 | 99.6 |
| Day 4 | 7.20 × 107 | 80,118 | 2.85 × 107 | 1,933 | 99.3 |
| Day 6 | 5.56 × 107 | 64,892 | 3.72 × 107 | 1,598 | 99.6 |
| Day 7 | 6.53 × 107 | 44,926 | 1.40 × 107 | 1,153 | 100 |
- aPercent decrease in ATP values vs. untreated organisms.
Identical P. carinii populations were tested as fresh isolates and after cryopreservation to compare viability over nine days in culture and sensitivity to pentamidine. In both populations, peak metabolic activity occurred from days 1 to 3. Cryopreserved organisms initially had lower ATP levels than fresh, but by day 7, achieved similar results. Cryopreserved organisms were 50% more sensitive to pentamidine 2 μ/ml at day 1 with sensitivities closer to freshly isolated organisms at all other time points.
The effect of host cell contamination on ATP readings was examined by enumeration of P.c. nuclei and host cells with HEMA-3 stained slides, and fluorescent labeling with ethidium homodimer-2 and calcein AM (Molecular Probes, Eugene, OR). Counts of host cells, both live and dead peaked at day 0 and declined throughout the 3-day assay period, while ATP levels increased to day 1 and remained stable thereafter.
Evaluation of sterol biosynthesis inhibitors [8], using Cryopreserved P. carinii populations showed these populations to be more sensitive to the sterol inhibitors than freshly prepared organisms. This was also the case when organisms were allowed to recover in media for 24-hours before test compounds were added. Cryopreserved organisms may be more sensitive to certain inhibitors such as those targeting sterols due to increased membrane biosynthesis as a result of cryopreservation.
The roles of nutrient depletion and contact inhibition were also evaluated in freshly isolated and Cryopreserved populations of P. carinii. Organisms were cultured at densities ranging from 1 × 106 to 1 × 109/ml, and were sampled for ATP content and fed daily for three days. When compared to the day 0 ATP readings, losses of up to 98% occurred in the most concentrated groups, while increases in ATP of up to 260% were observed at the lowest concentrations. No significant difference appeared between fresh and Cryopreserved populations, suggesting that they deplete the media of nutrients at essentially the same rate.
Conclusions
These experiments demonstrated that P. carinii maintained a suitable level of viability in cell free in vitro assay conditions after lengthy cryopreservation, and was responsive to standard anti-P. carinii compounds. Cryopreserved populations had enhanced sensitivity to some standard compounds such as pentamidine and sterol inhibitors. Enhanced sensitivity was not observed with compounds having no anti-P. carinii effects (e.g. ampicillin). P. carinii in culture, whether freshly processed or Cryopreserved, was extremely sensitive to population density. In our current culture conditions, 1 × 108 nuclei/ml is the maximum density at which viability can be suitably maintained.
Rat host cells were determined not to add significant amounts of ATP to the assay system. Host cell counts, both live and dead, decreased over the assay period, while P. carinii nuclei counts and ATP levels increased.
The benefits of using Cryopreserved and characterized organisms are numerous. An entire series of experiments can be conducted with organisms from a single donor; contaminated pools are screened out; and experiments can be conducted with genetically characterized organism populations. The final, and perhaps most significant benefit of these findings is the reduction of dependence on a consistent supply of rats with fulminant infections as an organism source, and thereby a reduction in the total number of animals needed to conduct in vitro screening studies.
ACKNOWLEDGMENTS
Supported by NIH grants RO1-AI-32426 and NO1-AI-75319.
