Axenic Culture of Schistosoma mansoni Sporocysts in Low O2 Environments Lia M. Bixler, Jennifer P. Lerner, Maria Ivanchenko, Rene´e S. McCormick, David W. Barnes*, and Christopher J. Bayne†, Department of Zoology and Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331; and *Division of Cell and Molecular Biology, American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110; †corresponding author. email: [email protected]
ABSTRACT: Recent successes in culturing intramolluscan larval stages of Schistosoma mansoni have relied on synxenic culture with a cell line (Bge) developed from embryos of a molluscan host Biomphalaria glabrata. To further facilitate progress toward control of schistosomiasis, a system for axenic in vitro culture of the parasite has now been developed. When culture media were preconditioned by Bge cells, sporocysts lived longer in vitro and produced more offspring. Because Bgederived components could be protecting sporocysts from oxidative stress, axenic sporocysts were cultured at lowered O2 levels. In an hypoxic environment, S. mansoni sporocysts grew well and produced daughter sporocysts continuously under axenic conditions and in a medium completely lacking host molecules. Sporocyst production occurs independently of host influence.
The human blood flukes Schistosoma mansoni, S. haematobium, S. japonicum, and S. mekongi cause nearly 200 million people worldwide to suffer the disease schistosomiasis and another 400 million to be at risk of contracting the disease (Bergquist and Colley, 1998). To produce cercariae (the larval stage that is infective for mammalian hosts), schistosome miracidia must first infect an intermediate host, in which they transform to mother sporocysts. In suitable hosts, these mother sporocysts grow and asexually produce daughter sporocysts. The snail Biomphalaria glabrata is an intermediate host of S. mansoni, and synxenic culture of sporocysts with a B. glabrata embryonic cell line (Bge) (ATCC CRL 1494) (Hansen, 1976; Bayne et al., 1978) has made it possible to propagate intramolluscan stages of the parasite (Yoshino and Laursen, 1995; Ivanchenko et al., 1999). Successful application of such synxenic cultures for the production of daughter sporocysts led us and others (Yoshino and Laursen, 1995) to examine whether media conditioned by Bge cells would support axenic development. However, longterm survival and reproduction of the parasites could not be achieved. Biomphalaria glabrata hemolymph has been reported to have an O2 level of 9% (Lee and Cheng, 1971, in Hansen, 1976). In earlier studies (Buecher et al., 1974; Hansen et al., 1974), daughter sporocysts were produced in axenic sporocyst cultures when CO2 and N2 were used to adjust O2 levels to 20%, 9%, 3%, and approximately 1%. With these lowered O2 levels and the addition of reducing compounds (dithiothreitol, free base cysteine, reduced glutathione), axenic sporocyst cultures produced daughters in media that had not been conditioned by other cells (Buecher et al., 1974). However, the daughter sporocysts produced under those conditions survived for only a few days. A method for
axenic culture of the intramolluscan stages of the parasite would greatly facilitate access to schistosome material for molecular, genetic, and pharmacological investigations. The authors have, therefore, further developed the in vitro culture of S. mansoni by combining the uses of (1) nitrogen to create a hypoxic environment and (2) previously optimized culture media. Using these conditions, a system has been developed in which the sporocysts thrive and reproduce for many generations, even in a medium free of host components. Schistosoma mansoni, PR1 strain, has been maintained in our lab since 1975, and male Syrian hamsters have served as the definitive host since 1992. M-line (Richards and Merritt, 1972) Biomphalaria glabrata have served as the molluscan intermediate hosts. To obtain miracidia (the larval stage that infects snails), eggs collected from 1 to 3 hamster livers are hatched under sterile conditions (Stibbs et al., 1979). Miracidia are transformed into sporocysts in Medium F base (Medium F lacking antibiotics and FBS; Stibbs et al., 1979) containing 20 mg/ml gentamicin (Life Technologies, Grand Island, New York) and 1% bovine serum albumin (Sigma A-3912; Sigma Chemicals, St. Louis, Missouri) (MFB-GB1). Typically, 10,000 to 30,000 miracidia are transformed overnight in 2 ml MFB-GB1 in a roller (0.1 rpm) at 26 C. The next day, sporocysts are washed in MFB-GB1 to free them of shed ciliated plates and are seeded into 24-well tissue culture plates (Corning, Inc., Corning, New York) at ca. 1,000 sporocysts per well. Sporocyst medium (SM) is composed of 23.75% Medium F base, 23.75% DMEM/F12 (Life Technologies) with 0.2 g/L NaHCO3 (Sigma Chemicals), 47.5% Bge medium (Hansen, 1976) containing 10% FBS (Hyclone, Logan, Utah) and 20 mg/ml gentamicin), 5% Serum Plus (JRH Biosciences, Lenexa, Kansas), 20 mg/ml gentamicin (in addition to the gentamicin in Bge medium), and 55 mM b-mercaptoethanol (Life Technologies). For preparation of conditioned sporocyst medium (CSM), the Bge medium component is first used for culture of Bge cells (1 to 3 days) and sterile-filtered to remove cells. SM and CSM are stored for up to 2 wk and 3 days, respectively, at 4 C. Since 1997, S. mansoni sporocysts have been cultured synxenically with Bge cells, maintained in a normoxic environment (approximately 21% O2) at 26 C, and fed with SM. At the time of this writing, sporocysts have continued to produce new generations for 20 mo in this system. With a single exception (due possibly to a particularly robust hatch of sporocysts), cultures held at atmospheric O2 levels and without Bge cells died within 1 wk, having produced no daughters. In initial attempts to culture sporocysts axenically in low O2 envi-
THE JOURNAL OF PARASITOLOGY, VOL. 87, NO. 5, October 2001
TABLE I. Number of daughter sporocysts produced by 1,000 sporocysts cultured axenically, counts taken on day 29 or 30.* Sporocysts
8.5% O2 SM CSM
1, 9, 3 28, 8, 14
0, 1, 1 22, 0, 0
18, 11, 17 9, 5, 26
Normoxic SM CSM
0, 0, 0 0, 0, 0
0, 0, 0 0, 0, 0
0, 0, 0 0, 0, 29
* SM, sporocyst medium; CSM, conditioned sporocyst medium.
ronments, portable Plexiglas incubator culture chambers (C.B.S. Scientific Company, Inc., Del Mar, California) were gassed with nitrogen, usually once or twice daily. Axenic sporocyst cultures maintained in these chambers produced daughters in 2 of 25 wells (8%) fed SM, and 18 of 87 wells (21%) fed CSM, indicative of beneficial effects of both low O2 and conditioning media with Bge cells. Of wells that produced daughters in incubator culture chambers, the numbers of daughters produced in a well of 1,000 sporocysts was less than 25 in half of the wells and no greater than 180 in any well, even when cultures were fed CSM. Because low O2 was found to be critical for production of daughters in axenic sporocyst cultures, the system used to establish and maintain hypoxic conditions was improved by means of an incubator (Model 3130; Forma Scientific, Marietta, Ohio) with an O2 regulator and connected to an N2 gas supply. Initially, with O2 set at 10%, it was noted that cultures appeared more healthy than those held in atmospheric O2, and they produced daughters. Subsequently, the O2 level was lowered to 6.0–8.5%. In experiments over 15 mo, axenic sporocyst cultures maintained at these O2 levels produced daughters in 92 of 97 wells (95%) when fed SM, and in 132 of 135 wells (98%) when fed CSM. The numbers of daughters also increased, with an average of approximately 200 daughters per well and a high value of 662 daughters in 1 well (all wells were seeded at ca. 1,000 sporocysts per well). In spite of the use of consistent procedures throughout their preparation, sporocysts derived from weekly hatches of miracidia have varied in their propensity to thrive. Production of daughters was therefore examined in groups of sporocysts within individual hatches. In each experiment, sporocysts from a single hatch were distributed into 2, 24well plates: 1 was placed in a normoxic incubator at 26 C, whereas the other was placed in an incubator set at 8.5% O2 and 26 C. Each plate contained 6 wells of sporocysts, seeded at 1,000 sporocysts/well; 3 wells were fed SM, and 3 wells were fed CSM. In all 3 experiments (Table I), the number of wells in which daughters were produced and the numbers of daughters produced per well were below the 15-mo average. As noted, sporocyst vigor and, therefore, daughter production varied from 1 hatch to another; however, several facts are clear: normoxic conditions are deleterious to sporocysts, preconditioning of the Bge component of the medium is beneficial to sporocysts, and variance is high. Incubator culture chambers aid in the production of daughters from axenic S. mansoni sporocyst cultures, and they are less expensive than incubators with O2 sensors and gas flow regulators. However, they need to be regassed each time they are opened, and they do not provide constant hypoxic environments. When 1 culture chamber was gassed no more than once every 3 days, none of the cultures in it produced daughters, probably because of an imperfect hermetic seal. Sporocyst daughter production is consistently and markedly increased with the use of a more controlled environment, such as that provided by an incubator with an O2 sensor and N2 gas supply. Under normoxic conditions, axenic S. mansoni sporocysts do not produce daughters when fed SM, whereas daughters have been produced in 2 of 48 wells (4%) fed CSM. In a low (but variable) O2 environment provided by incubator culture chambers, axenic sporocyst cultures are more likely to produce daughters when fed CSM
than SM. However, when O2 is held at a constant 8.5% by a controlled gas incubator, any beneficial effects of conditioned media are less evident: at 8.5% O2, axenic sporocyst cultures are nearly as likely to produce daughters whether fed CSM or SM. This implies that CSM may contain antioxidants that protect sporocysts from oxidative damage that occurs at higher O2 levels. It is suggested that in a wellcontrolled hypoxic environment, the lowered O2 levels are so beneficial for sporocyst health and daughter production that the putative antioxidant activity due to Bge cell conditioning of the medium is less essential to axenic sporocysts. When the O2 level of the incubator was reduced to 6.0%, all cultures continued to proliferate, as some have for over a year. An initial wave of small, bright daughter sporocysts usually appears after 3 to 4 wk; later on, synchrony is lost so that generation times are less easily tracked. However, small, bright daughters continue to appear in wells as long as cultures are maintained. The most significant finding reported here is that it is possible to produce daughter sporocysts in media that are completely free of host components. Such components were essential to ensure daughter sporocyst production only under normoxic conditions. Oxidants arising in such cultures, either spontaneously because of high oxygen tensions or from the metabolic actions of the axenic sporocysts themselves, may be toxic. Lower oxygen pressures probably prevent the formation of these toxic oxidants, and the addition of b-mercaptoethanol to the medium (pers. obs.) benefits sporocyst cultures, probably by reducing reactive oxidants. Cercariae were produced earlier in vitro (Ivanchenko et al., 1999) in sporocysts cultured with Bge cells. Cercariogenesis has not been observed in axenic cultures, and its trigger remains elusive. It is likely to require one or more host components. This research was supported by NIH grants AI-16137 and RR-12063 and by the United Nations Development Program/World Bank/World Health Organization Program for Research and Training in Tropical Diseases. LITERATURE CITED BAYNE, C. J., A. OWCZARZAK, AND J. R. ALLEN. 1978. Molluscan (Biomphalaria) cell line: Serology, karyotype, behavioral, and enzyme electrophoretic characterization. Journal of Invertebrate Pathology 32: 35–39. BERGQUIST, N. W., AND D. G. COLLEY. 1998. Schistosomiasis vaccines: Research to development. Parasitology Today 14: 99–104. BUECHER, E. J., G. PEREZ-MENDEZ, E. L. HANSEN, AND E. YARWOOD. 1974. Sulfhydryl compounds under controlled gas in culture of Schistosoma mansoni sporocysts. Proceedings of the Society for Experimental Biology and Medicine 146: 1101–1105. HANSEN, E. L. 1976. A cell line from embryos of Biomphalaria glabrata (Pulmonata): Establishment and characteristics. In Invertebrate tissue culture, E. Kurstak and K. Maramorosch (eds.). Academic Press, New York, New York, p. 75–99. ———, G. PEREZ-MENDEZ, E. YARWOOD, AND E. J. BUECHER. 1974. Second-generation daughter sporocysts of Schistosoma mansoni in axenic culture. Journal of Parasitology 60: 371–372. IVANCHENKO, M. G., J. P. LERNER, R. S. MCCORMICK, A. TOUMADJE, B. ALLEN, K. FISCHER, O. HEDSTROM, A. HELMRICH, D. W. BARNES, AND C. J. BAYNE. 1999. Continuous in vitro propagation and differentiation of cultures of the intramolluscan stages of the human parasite Schistosoma mansoni. Proceedings of the National Academy of Sciences, USA 96: 4965–4970. LEE, F. O., AND T. C. CHENG. 1971. Schistosoma mansoni: Respirometric and partial pressure studies in infected Biomphalaria glabrata. Experimental Parasitology 30: 393–399. RICHARDS, C. S., AND J. W. MERRITT. 1972. Genetic factors in the susceptibility of juvenile Biomphalaria glabrata to Schistosoma mansoni infection. American Journal of Tropical Medicine and Hygiene 21: 425–443. STIBBS, H. H., A. OWCZARZAK, C. J. BAYNE, AND P. DEWAN. 1979. Schistosome sporocyst-killing amoebae isolated from Biomphalaria glabrata. Journal of Invertebrate Pathology 33: 159–170. YOSHINO, T. P., AND J. R. LAURSEN. 1995. Production of Schistosoma mansoni daughter sporocysts from mother sporocysts maintained in synxenic culture with Biomphalaria glabrata embryonic (Bge) cells. Journal of Parasitology 81: 714–722.