Long-Term Culture of Giardia lamblia in Cell Culture Medium Requires Intimate Association with Viable Mammalian Cells

Giardia lamblia is usually cultured axenically in TYI-S-33, a complex medium which does not permit survival and growth of mammalian cells. Likewise, medium commonly used to maintain and grow mammalian cells does not support healthy trophozoite survival for more than a few hours. The inability to coculture trophozoites and epithelial cells under optimal conditions limits studies of their interactions as well as interpretation of results.

ABSTRACT

Giardia lamblia is usually cultured axenically in TYI-S-33, a complex medium which does not permit survival and growth of mammalian cells. Likewise, medium commonly used to maintain and grow mammalian cells does not support healthy trophozoite survival for more than a few hours. The inability to coculture trophozoites and epithelial cells under optimal conditions limits studies of their interactions as well as interpretation of results. Trophozoites of the WB isolate but not the GS isolate were repeatedly adapted to grow stably in long-term cocultures with Caco2, Cos7, and mouse tumor rectal (RIT) cell lines using hybridoma-screened Dulbecco’s modified Eagle’s medium and 10% fetal calf serum. Giardia did not grow in spent cell culture medium or when separated by a permeable membrane using transwell methodology. Giardia chronically cocultured with specific cell lines became adapted (conditioned). These Giardia cocultures grew better than nonconditioned trophozoites, and the cell lines differed in their ability to support trophozoite growth in the order of RIT > Cos7 > Caco2. Trophozoites conditioned on one cell line and then grown in the presence of a heterologous cell line changed their growth rate to that seen in conditioned Giardia from the heterologous cell line. Trophozoite survival required intimate contact with cells, suggesting that trophozoites obtain an essential nutrient or growth factor from mammalian cells. This may explain why Giardia trophozoites adhere to the small intestinal epithelium during human and animal infections. This coculture system will be useful to understand the complex interactions between the host cells and parasite.

INTRODUCTION

Giardia lamblia, a common gastrointestinal parasite of humans and animals, is usually cultured axenically in a complex medium (TYI-S-33) (1), which is toxic to mammalian cells and cultured mammalian cell lines (MCLs). Although Giardia trophozoites show no observable morphological changes for a limited time in cell culture media, after a few hours their cellular integrity becomes altered, and by 24 h most have died or are degenerating (2 and personal observations). Inability to maintain viable host cells and Giardia together for significant lengths of time has limited studies of interactions between host and parasite and may cloud the interpretation of previous findings.

During unrelated experiments to detect molecular interactions between Giardia trophozoites and intestinal cells, trophozoites were cultured with monolayers of MCLs for a limited time. As expected, after 24 h most trophozoites had died, but surprisingly, a few trophozoites had adhered to the cell monolayer or onto glass surfaces in close proximity to cells and demonstrated typical mobility. Using this and similar coculture approaches, we were able to stably grow trophozoites in coculture with MCLs in standard cell culture medium (CCM). Here, we describe the methods, requirements, and biological implications of long-term cultivation of Giardia with MCLs.

RESULTS

Culture of Giardia trophozoite with mammalian cells.

During unrelated experiments to study molecular interactions between trophozoites and mammalian cells, trophozoites were allowed to adhere to a monolayer of mammalian cells and observed over the next 24 h. The initial observation showed that after adding WB isolate trophozoites to Caco2 cells, almost all the trophozoites died. However, a few morphologically normal motile trophozoites had adhered to Caco2 cells or to the glass tube in close proximity to cells. Over time and with judicious medium replacement and careful attention to the viability of cells and Giardia, both the mammalian cells and trophozoites survived and grew. These results were reliably reproduced using Caco2 cells, Cos7 cells, and mouse rectal tumor cells (RIT). All 3 MCLs were able to support sustained trophozoite growth of naive nonconditioned WB Giardia, which readily adapted to grow in the presence of MCL in CCM (Fig. 1).

FIG 1.

Pictures of trophozoites cultured with RIT cells at moderate (A) and increased density of trophozoites (B) and trophozoites with Cos7 cells (C) and Caco2 cells (D). The preferential association of trophozoites with cells is clearly seen in panels A and C.

There was moderate variability of trophozoite growth in replicate experiments. Twelve 13-ml tubes previously seeded with Cos7 cells and allowed to form monolayers were inoculated with 9,400/ml trophozoites, and the number of trophozoites was determined over 6 days. On day 2 the number increased to 12,167 ± 4,130/ml and on day 6 to 32,333 ± 14,112/ml trophozoites (means ± standard deviations [SD]) (P < 0.001). The growth rate was much lower than that for WB Giardia maintained in TYI-S-33 medium, which has a doubling time in our laboratory of about 6 to 8 h. Trophozoites with all 3 MCLs were passaged stably for over a year and did not lose their ability to grow in TYI-S-33.

Coculture requirements.

We were unable to grow trophozoites in CCM in the absence of close contact with viable mammalian cells. Careful attention to refeeding medium to maintain healthy and nonacidic monolayers was essential for trophozoites to survive and multiply. Mammalian cell lines that grew faster than trophozoites overgrew and degenerated; subsequently, the trophozoites degenerated, did not grow, and at times could not be rescued. Observations suggested the best combinations were when trophozoites and mammalian cells grew at approximately the same rate. WB trophozoites did not survive when inoculated into 2-week-old spent medium harvested from Caco2 cells, suggesting that direct or close association with viable cells was required. Alternative explanations are that one or more essential ingredients were either labile or of insufficient concentration in spent medium.

The need for direct or close contact was further explored using transwell methodology. In multiple attempts trophozoites could not be stably maintained, although some organisms survived for more than 10 days before dying off. This longer-than-expected survival suggested partial growth enhancement but ultimate inability to support sustained growth. These experiments were complicated by the inability of some cell lines to survive under the anaerobic conditions produced by the Bio-Bag anaerobic generating system.

The ability for mammalian cells to support trophozoites was isolate dependent. Numerous attempts to culture isolate GS with MCLs were unsuccessful.

Comparison of growth of conditioned and nonconditioned trophozoites.

Trophozoites that had been stably established in cell culture (conditioned Giardia) grew without the usual die-off in numbers seen when cultures were first established and seemed to become adapted to grow with mammalian cell lines. Figure 2 shows that Cos7 and RIT trophozoites conditioned for 8 months grew significantly better than nonconditioned trophozoites on the same cell lines, and conditioned Caco2 trophozoites grew somewhat (P > 0.05) faster than nonconditioned trophozoites. Nonconditioned Cos7 (P < 0.02 by nonpaired t test) and RIT trophozoites (P = 0.01) grew significantly over 4 days, but the increase in nonconditioned Caco2 trophozoites did not reach significance (P > 0.05). RIT conditioned and nonconditioned trophozoites grew significantly better (P < 0.05) than their similarly grown Cos7 and Caco2 trophozoite counterparts; Cos7 trophozoites grew nonsignificantly better than Caco2 trophozoites (P > 0.05).

FIG 2.

Growth of conditioned and nonconditioned Giardia trophozoites over time cocultured with 3 mammalian cell types. Results are shown as means ± standard errors (SE). The growth of naive nonconditioned trophozoites and growth of conditioned trophozoites that had been cocultured with the 3 MCLs over 8 months were compared in cocultures. Although the number of nonconditioned trophozoites increased over time, the growth of conditioned Cos7 and RIT trophozoites increased significantly compared to levels for nonconditioned trophozoites. Conditioned RIT trophozoites grew best. See the text for fuller description and statistical analysis.

Growth of MLCs with conditioned and nonconditioned trophozoites.

Although the relationship between trophozoite growth and MCL growth varied somewhat between experiments, the results of multiple experiments showed that RIT cells grew fastest, reached greater cell numbers, and allowed trophozoites to grow to greater numbers than the other MCLs (Fig. 3). Cos7 cells grew less well than RIT cells and supported trophozoites less well than RIT cells but better than Caco2 cells. The latter grew poorly and did not initially support Giardia growth well, but over time trophozoite growth improved. In one experiment comparing the growth of MCLs with either homologous conditioned or nonconditioned trophozoites, MCLs grew better with conditioned than nonconditioned trophozoites and reached significance in most time periods. In particular, at the end of the experiment on day 4, Caco2 cells in the presence of conditioned Caco2 Giardia reached the same number as Cos7 cells, something not seen using nonconditioned Giardia.

FIG 3.

Growth of 3 different cell lines cocultured with homologous conditioned or nonconditioned Giardia trophozoites over time. Results are shown as means ± SE. The numbers of MCLs were significantly greater in conditioned cultures than nonconditioned cultures at days 2 and 4 for Caco2, days 1 to 3 for Cos7, and days 2 to 3 for RIT cells (P < 0.05).

Growth of trophozoites with heterologous mammalian cell lines.

The specificity of the conditioned Giardia for its homologous cell culture was determined by culturing Cos7 and RIT conditioned Giardia with heterologous mammalian cells (Fig. 4). Cos7 conditioned Giardia grew significantly better with RIT cells than homologous Cos7 cells (P = 0.011), and RIT conditioned Giardia grew significantly worse than with its homologous cell culture (P = 0.045). The trophozoites did not undergo the usual die-off seen with nonconditioned trophozoites, suggesting that the cell lines were providing the same benefits but in different amounts or combinations.

FIG 4.

Comparison of conditioned Cos7 and RIT trophozoites on homologous and heterologous MCLs. Cos7 conditioned Giardia grew significantly better with RIT cells than homologous Cos7 cells (P = 0.011), and RIT conditioned Giardia grew significantly worse than with its homologous cell culture (P = 0.045).

DISCUSSION

TYI-I-33 is the standard medium to grow Giardia, but this medium does not support survival or growth of mammalian cells. We show that Giardia can survive and grow stably in various types of cell lines, which should allow more exacting and prolonged studies of the effects of Giardia on intestinal cells and perhaps immune cells. We were unable to grow trophozoites without intimate association with cells. Different cell lines supported Giardia growth and survival to various degrees, which may be due in part to disparity in growth rates between Giardia and cell lines. Only the prototypic group 1 (3–5) or assemblage A isolate WB and not isolate GS, a prototypic group 3 (3–5) or assemblage B isolate, was able to grow stably in the MCL tested. Other isolates were not tested.

The major findings are the ability to grow Giardia and mammalian cells together indefinitely, and this allows the study of the interaction of trophozoites and mammalian cells. Giardia trophozoites become adapted to grow with cells, and different cell types support trophozoite growth to various degrees. Trophozoites required intimate or direct contact with cells to survive. Lastly, cell lines continued to grow in the presence of conditioned trophozoites. The mechanisms involved in these observed changes are unknown and likely complex. This system allows a way to study cell and trophozoite changes and to understand these adaptions on a molecular level.

Fisher et al. (6) performed experiments and were able to grow Giardia in the presence of Caco2 cells with 10% TYI-S-33 and Dulbecco’s modified Eagle’s medium (DMEM) for up to 21 days in a transwell system. The purpose and design of their experiments were to study the interaction of Giardia with Caco2 cells, which contrasts with the biological questions studied and reported here. In their experiments, the addition of Giardia medium was necessary but may have altered the character of the interaction. In unreported results, we added limited increased amounts of bile and cysteine (essential components of TYI-S-33) to CCM, and although Giardia growth increased and MCLs survived, Giardia was less associated with cells on visual inspection; therefore, use of these additives was not studied further.

Exactly how Giardia adapts to culture conditions has not been studied. Axenizing Giardia isolates in TYI-S-33 is an uncertain endeavor. Initial growth of Giardia during the axenization process is commonly poor, but eventually organisms adapt to the medium. However, a sizable but unclear number cannot be axenized and never adapt. Use of mammalian cell lines might be an easier or more efficient way to grow Giardia when first established in vitro. Similarly, Giardia grown in TYI-S-33 subjected to CCM does not grow well initially but does adapt. However, even then, it grows slowly compared to growth in TYI-S-33.

The above-described experiments were performed about 15 years ago. Numerous studies prior to and succeeding these experiments have shown deleterious changes of Giardia on intestinal cell cultures in short-term experiments (7–9). Our studies were not designed to determine the effects of Giardia trophozoites on cells. However, it is of interest that, paradoxically, instead of observing expected deleterious effects of Giardia on cells, preliminary experiments suggested that overall, MCLs grow better when cultured in the presence of conditioned trophozoites than nonconditioned trophozoites. This may be due to the deleterious effects of degenerating nonconditioned trophozoites on cells, which did not occur with conditioned trophozoites.

On the other hand, perhaps there is a cell growth-promoting effect of these conditioned organisms, which unfortunately was not tested directly since there was no comparison of cell growth without Giardia. However, the lack of deleterious effects of conditioned Giardia on cells is consistent with asymptomatic carriage and the accommodative course of chronic infections in the tropical regions where asymptomatic Giardia infections are the rule.

Besides the development of a useful cocultivation system, an important observation was the apparent essential requirement for intimate and/or close association of trophozoites with cells, which may be the reason why Giardia trophozoites adhere to the small intestinal mucosa during infection. MCLs may supply something essential for growth, such as a required nutrient or growth factor. This culture system can be used to better dissect cell-trophozoite interactions.

MATERIALS AND METHODS

The WB isolate (10) was used exclusively, with the exception of attempts to establish cocultures of isolate GS (3) with MCLs. Trophozoites were maintained in TYI-S-33 medium as previously reported (1), harvested for use by centrifugation, and suspended in CCM for inoculation into MCL monolayers. Human epithelial colorectal cells (Caco2; ATCC HTB-37), African green monkey kidney fibroblast-like cells (Cos7; ATCC 1651), and mouse tumor rectal cells of unclear origin were maintained in tissue culture flasks in Dulbecco’s modified Eagle’s medium with glucose (DMEM; lot 016104; BioWhittaker, Walkersville, MD) (CCM) and 10% heat-inactivated hybridoma-screened fetal calf serum (FCS; lot 016371; BioWhittaker) (11). In initial studies, monolayers of adhered mammalian cells were established in 13-ml glass tubes normally used to grow Giardia and maintained at 37°C in medium-filled tubes. Giardia trophozoites were suspended in CCM and added to the glass tubes containing MCL monolayers, and the tubes were fully refilled. Trophozoites and cells were visualized using an inverted microscope optimized to view adhered cells on curved glass tubes. Various culture vessels, including 8-ml glass tubes, were used, but because of the ease of visualization and use, most experiments and long-term cultures employed Nunclon air-tight capped, flat-sided, polystyrene 10-ml culture tubes (ThermoFisher Scientific, Waltham, MA). Medium was changed by fully or partially removing spent medium and replacing it with fresh CCM when growth or morphology of the mammalian cells or Giardia deteriorated or to prevent cell overgrowth and development of acidic medium.

Usual experiments employed triplicate flat-bottom tubes for each time point. Tubes were held at 37°C with CCM almost but not completely filling the tube. Monolayers were established with either 1 × 10⁴ Cos2 and RIT cells or 2 × 10⁴ Caco2 cells and left to grow for about 5 to 6 days until 60 to 100% confluence. After adding fresh medium, tubes were inoculated with 10.5 × 10⁴/ml trophozoites (105,000/tube) and cultured at 37°C in a closed system, and counts were determined as detailed below. Medium was changed similarly for all tubes at the same time and in the same manner.

To count the number of trophozoites and mammalian cells in tubes over time, triplicate tubes were cooled on ice for 20 min, and then the medium containing detached Giardia and loose cells was decanted into ice-cold tubes for counting using a hemocytometer. In the next step, 2 ml of 0.25% trypsin-EDTA (ThermoFisher, Waltham, MA) was added to detach the monolayers and residual Giardia. The tubes were then agitated on a rotary shaker at 4°C for 20 min, cold phosphate-buffered saline was added up to the original 10.5-ml volume, the solution was vigorously mixed, and a sample was taken for cell and trophozoite counts. An occasional trophozoite was detected in dispersed mammalian cells and added to the number counted in its prior decanted supernatant.

Growth of Giardia chronically maintained with Caco2, Cos7, and mouse tumor rectal cells for 8 months (conditioned Giardia) was compared to growth of the same WB Giardia culture passaged in TYI-S-33. Procedures were similar to those described above. The numbers of viable trophozoites and mammalian cells were determined over the next 4 days.

The ability of MCLs to support heterologous conditioned Giardia was tested by growing conditioned Cos7 trophozoites with RIT cells and conditioned RIT trophozoites with Cos7 cells and compared to trophozoites grown with homologous cell cultures.

To determine if trophozoites required direct contact with mammalian cells for growth, Giardia and Cos7, Caco2, and RIT cells were inoculated onto transmembrane Costar 3450 transwells (24-mm diameter, 0.4-μm-pore-size polyester membrane; Corning, Tewksbury, MA) placed into culture plates with 2.5-ml wells. MCLs were placed either into the top of the transwell insert or into the well and allowed to form a monolayer. A total of 10,000 Giardia organisms/ml were then added to the compartment on the opposite side of the membrane and cultured in Bio-Bags (Bio-Bag environmental chamber type A; Marion Scientific, KS) anaerobically and aerobically as described previously (12), and the viability and state of the cells and trophozoites were determined visually over time.

Statistical analyses.

Means were used throughout as measures of central tendency. Student's t test was used to determine statistical differences.