Application of a MTT assay for screening nutritional factors in growth media of primary sponge cell culture

Marine sponges (Porifera) are producers of the largest variety of bioactive compounds among benthic marine organisms. In vitro culture of marine sponge cells has been proposed for the sustainable production of these pharmacologically interesting compounds from marine sponges but with limited success. The development of a suitable growth medium is an essential prerequisite for sponge cells grown in vitro. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was adapted to screen for potential nutritional factors in formulating a growth medium for primary cell culture of Suberites domuncula. In 96-well plates, the effects of nutritional factors including glutamine, pyruvate, iron citrate, silicon, RPMI 1640, and Marine Broth 2216 on the viable cell density were examined in primary cell culture of S. domuncula 36 h after inoculation. Ferric iron (Fe(3+)) and pyruvate were found to significantly improve cell viability in a dose-dependent manner. Silicon and glutamine showed limited improvements at certain concentrations. The supplement of RPMI 1640 and Marine Broth 2216 did not increase cell viability. As a result, several improved media able to maintain higher cell viability in a short-term culture of primary sponge cells could be formulated.     

Sustained growth of explants from Mediterranean sponge Crambe crambe cultured in vitro with enriched RPMI 1640

Marine sponges are potential sources of many unique metabolites, including cytotoxic and anticancer compounds. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. It is commonly accepted that tissue (fragments, explants, and primmorphs) and in vitro cell cultivation show great potential. However, there is little knowledge of the nutritional requirements of marine sponges to carry out efficient and sustained in vitro culture and progress has been slow. In marine invertebrate fila many unsuccessful attempts have been made with in vitro cultures using typical commercial animal cell media based on sources of dissolved organic carbon (DOC) (e.g., DMEM, RPMI, M199, L-15, etc.). One of the reasons for this failure is the use of hardly identifiable growth promoters, based on terrestrial animal sera. An alternative is the use of extracts from marine animals, since they may contain nutrients necessary for growth. In this work we have cultivated in vitro explants of the encrusting marine sponge Crambe crambe. It is one of the most abundant sponges on the Mediterranean coastline and also possesses an array of potentially active metabolites (crambines and crambescidins). Initially a new approach was developed in order to show consumption of DOC by explants. Thus, different initial DOC concentrations (300, 400, 700 and 1200 mg DOC L(-1)) were assayed. Consumption was evident in all four assays and was more marked in the first 6 h. The DOC assimilation data were adjusted to an empirical model widely used for uptake kinetics of organic dissolved compounds in marine invertebrates. Second, a protocol was established to cultivate explants in vitro. Different medium formulations based on RPMI 1640 commercial medium enriched with amino acids and inorganic salts to emulate seawater salinity were assayed. The enrichment of this medium with an Octopus aqueous extract in the proportions of 10% and 20% (v/v) resulted in an evident sustained long-term growth of C. crambe explants. This growth enhancement produced high metabolic activity in the explants, as is confirmed by the high ammonium and lactate content in the medium a few days after its renewal and by the consumption of glucose. The lactate accumulation increased with the size and age of explants. Prior to these experiments, we successfully developed a robust new alternative method, based on digital image treatment, for accurate determination of the explant apparent volume as growth measure.     

Characterization of porous poly(D,L-lactic-co-glycolic acid) sponges fabricated by supercritical CO2 gas-foaming method as a scaffold for three-dimensional growth of Hep3B cells

This study presents the application of the porous poly(D,L-lactic-co-glycolic acid) (PLGA) sponges fabricated from an organic solvent free supercritical gas foaming technique. Two formulations of PLGA sponges with different co-polymer compositions (85:15 and 50:50) were fabricated as novel scaffolds to guide human hepatoma cell line, Hep3B cell growth in vitro. The PLGA sponges showed desirable biodegradability and exhibited uniform pore size distribution with moderate interconnectivity. It was observed in this study that cells cultured on PLGA sponges showed lower proliferation rate as compared to the control during 14 days of culture as measured by using total DNA and methylthiazol tetrazolium (MTT) assays. However, the cells cultured on the sponges tended to aggregate to form cell islets which were able to express better hepatic functions. The enzyme-linked immunosorbent assay (ELISA) results showed that the cell-sponge constructs secreted 1.5-3.0 times more albumin than the control when normalized to cellular content. In a similar fashion, its detoxification ability was also predominantly higher than that of the control as indicated by the ethoxyresorufin-O-deethylase (EROD) results. By comparing the cells growing on the two formulations of PLGA sponges, it was found that the PLGA 85:15 sponge exhibited better conductive and desirable environment for hep3B cells as justified by better cell infiltration, higher proliferation and hepatic function than the PLGA 50:50 sponge.     

Formulation of a basal medium for primary cell culture of the marine sponge Hymeniacidon perleve

Marine sponge cell culture is a potential route for the sustainable production of sponge-derived bioproducts. Development of a basal culture medium is a prerequisite for the attachment, spreading, and growth of sponge cells in vitro. With the limited knowledge available on nutrient requirements for sponge cells, a series of statistical experimental designs has been employed to screen and optimize the critical nutrient components including inorganic salts (ferric ion, zinc ion, silicate, and NaCl), amino acids (glycine, glutamine, and aspartic acid), sugars (glucose, sorbitol, and sodium pyruvate), vitamin C, and mammalian cell medium (DMEM and RPMI 1640) using MTT assay in 96-well plates. The marine sponge Hymeniacidon perleve was used as a model system. Plackett-Burman design was used for the initial screening, which identified the significant factors of ferric ion, NaCl, and vitamin C. These three factors were selected for further optimization by Uniform Design and Response Surface Methodology (RSM), respectively. A basal medium was finally established, which supported an over 100% increase in viability of sponge cells.     

Search for improved culture conditions for clonogenic growth of human colorectal cancer cells in vitro

In order to analyze and define potentially better growth conditions for colonic stem cell proliferation, we chose four established human colorectal cancer cell lines that differed in biologic cell properties. We studied variables of standard cloning conditions including culture medium, serum supplement, solidifying agent, addition of specific growth factors and use of capillaries as an alternative culture vessel. While modulation of serum concentration as well as use of various standard formulations of culture base media did not result in a reproducible increase of plating efficiencies (PEs), a significant increase in colony formation (when compared to the conventional assay procedure) was achieved; by use of 0.3% agarose or boiled agar as semisolid matrix and by culturing of cells in enriched 'GMF medium'. Specific growth factors, such as EGF or glucagon resulted in "occasionally better" in vitro growth. This suggests a retention of the ability of cells in culture to respond to physiologic regulators of growth. To verify and extend these initial results obtained with continuous cell lines, growth enhancing modifications of the original cloning technique were subsequently applied to in vitro growth of 15 human colorectal cancer specimens obtained directly from patients. Specimens that grew 30 or more colonies under standard plating conditions displayed a more than two-fold increase in PEs which was reproducible for the two specific variables mentioned above, but the overall success rate of the assay could not be improved. In addition to the possibility that several deficient basic requirements for achieving optimal environmental conditions for colonic stem cell growth have not been defined, we believe a major reason for failing to improve the number of drug-assayable specimens is related to an inherent interneoplastic diversity in terms of growth requirements of human colorectal malignancies.     

Development in primary cell culture of demosponges

We have established primary cell culture of the marine demosponge Dysidea avara and Suberites domuncula. Microbial contamination was controlled by the use of a pool of antibiotics confirming the goodness of this procedure. Effect of pH, temperature and light was studied to establish the better growth conditions. The comparison of lipid composition of sponge and cells suggested a series of experiments to optimise the medium. A glucose dose-dependent experiment showed that the ideal glucose concentration is 1 g l(-1). Supplementing the medium with unsaturated fatty acid and retinol, no promotion of growth was observed, but the compounds were totally metabolised by cells. Increments from 70 to 160% in the number of cells were observed, supplementing the medium with different concentration of cholesterol. These results suggest that the analysis of the chemical composition of sponge and cells give indication on the composition of the nutrient media.     

Characteristics of batch culture of a recombinant bacillus brevis excreting a foreign esterase

The productivity of extracellular enzyme was evaluated in batch culture using a protein hyperexcreting host, Bacillus brevis HPD31(5) harboring pHSC131, which carried a gene (est) encoding esterase activity from Bacillus stearother mophilus. Optimum temperature and pH for the bacterial growth and the production of extracellular esterase were found to be 35 degrees C and pH 6.5, by using the standard medium (GPY) containing neomycin as a selective pressure, Under the cultivation condition employed, cell growth reached 5 g dry cell weight/L, while the extracellular esterase activity amounted to 4.5 U/mL. Most (79%-92%) of the esterase produced was excreted into the medium. pHSC131 was stably retained in the host cell during cultivation in the presence of neomycin. However, in the absence of neomycin, the plasmid was completely lost from the host after 12-h cultivation accompanied by decreases in both esterase activity and production of total extracellular protein. The copy number of the plasmid was estimated to be approximately 7 throughout the cultivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the excreted proteins showed the presence of a protein having an apparent molecular weight of 32,000, which equals to the value predicted from the DNA sequence of the est gene.     

A covert contaminant of cultured plant cells: elimination of a Hyphomicrobium sp. from cultures of Datura innoxia (Mill.)

We have discovered a bacterial contaminant in some cell cultures of Datura innoxia (Mill.). The bacterium was tentatively identified as a species of Hyphomicrobium on the basis of its morphology and life cycle, and was isolated and grown in pure culture on a defined medium. The contaminant was not macroscopically observable in plant cell cultures. It caused neither a reduction of plant cell growth nor a noticeable increase in culture turbidity. Furthermore, it was not readily detectable by many standard assays for culture contamination: it would not grow alone in plant culture medium or yeast extract potato dextrose medium, and grew only very slowly on nutrient agar or beef-peptone medium. Repeated treatments with a combination of streptomycin (100 g/ml) and carbenicillin (100 g/ml) eliminated the contaminant from D. innoxia cell cultures without harming the plant cells.     

Cell cycle analysis of primary sponge cell cultures

Proliferation of sponge cells is generally measured via cell counts or viability assays. However, more insight into the proliferative state of a sponge cell population can be obtained from the distribution of the cells over the different phases of the cell cycle. Cell cycle distribution of sponge cells was measured via flow cytometry after staining the DNA with propidium iodide. The five sponges studied in this paper all showed a large fraction of cells in G1/G0 compared to G2/M and S, indicating that cells were not actively dividing. In addition, some sponges also showed a large apoptotic fraction, indicating cell death. Additional apoptosis measurements, based on caspase activity, showed that harvesting and dissociation of sponge tissue to initiate a primary cell culture was directly correlated with an increase in apoptotic cells. This indicates that for the development of cell cultures, more attention should be given to harvesting, dissociation, and quality of starting material. Finally, cultivation conditions used were ineffective for proliferation, since after 2 d of cultivating Haliclona oculata cells, most cells shifted towards the apoptotic fraction, indicating that cells were dying. For development of in vitro sponge cell cultures, flow cytometric cell cycle analysis is a useful method to assess the proliferative state of a sponge cell culture and can be used to validate improvements in harvesting and dissociation, to select sponges with good proliferative capacities and to study the influence of culture conditions for stimulating cell growth.     

Bioproduction of antimicrobial compounds by using marine filamentous cyanobacterium cultivation

The synthesis of bioactive compounds with antimicrobial activity, excreted by marine cyanobacteria, strongly depends on their growth conditions. Due to the wide variety of biomolecules which could show properties as growth inhibitors and their low concentrations within the culture medium, the activity of their crude extracts also seems to be related to the extraction method used. Using the marine filamentous cyanobacterium Geitlerinema strain Flo1, we demonstrate a systematic approach for identifying optimal culture conditions to obtain culture media extracts with antimicrobial activity. The changes in the culture conditions, such as the addition of NaBr to the medium, cell immobilisation in vegetable sponge pieces, or temperature, effected the production of these bioactive compounds. The crude extract, containing middle polar molecules, obtained by extraction with Amberlite XAD-1180 had a higher antifouling activity upon a number of bacteria and fungi than the extract obtained by extraction with Amberlite XAD-16. The lowest inhibitory concentration obtained upon Rhodosporidium sphaerocarpum was still ten times higher than that of bis(tributyltin)oxide, but compared to zinc pyrithione, it was two times more active.     

Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells

This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing beta-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both beta-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies.     

Efforts to develop a cultured sponge cell line: revisiting an intractable problem

Residents of the marine environment, sponges (Porifera) have the ability to produce organic compounds known as secondary metabolites, which are not directly involved in the normal growth, development, or reproduction of an organism. Because of their sessile nature, the production of these bioactive compounds has been interpreted as a functional adaptation to serve in an important survival role as a means to counter various environmental stress factors such as predation, overgrowth by fouling organisms, or competition for limited space. Regardless of the reasons for this adaptation, a variety of isolated compounds have already proven to demonstrate remarkable anticancer, fungicidal, and antibiotic properties. A major obstacle to the isolation and production of novel compounds from sponges is the lack of a large, reliable source of sponge material. Sponge collection from the sea would be environmentally detrimental to the already stressed and sparse sponge populations. Sponge production in an aquaculture setting might appear to be an ideal alternative but would also be cost-ineffective and sponge growth is extremely slow. A third approach involves the development of a sponge cell culture system capable of producing the necessary cell numbers to harvest for research purposes as well as for the eventual commercial-scale production of promising bioactive compounds. Unfortunately, little progress has been made in this direction other than the establishment of temporary cultures containing aggregates and fragments of cells. One impediment toward successful sponge cell culture might be ascribed to a lack of published knowledge of failed methodologies, and thus, time and effort is wasted on continued reinvention of the same methods and procedures. Consequently, we have undertaken here to chart some of our unsuccessful research efforts, our methodology, and results to provide the sponge research community with knowledge to assist them to better avoid taking the same failed pathways.     

Biphasic calcium response of platelet-derived growth factor stimulated glioblastoma cells is a function of cell confluence.

BACKGROUND: Previous reports have linked the spiking or two-phased character of calcium transients evoked by platelet-derived growth factor (PDGF) to the position of cells in the cell cycle without regard to cell-cell contact and communication. Because cell confluence can regulate growth factor receptor expression and dephosphorylation, we investigated the effect of cell culture confluence and cell cycle on calcium responses of PDGF-BB-stimulated A172 glioblastoma cells. METHODS: Digital imaging cytometry was used to correlate the peak and duration of calcium response with bromodeoxyuridine positivity and DNA content and with culture confluence on a cell-by-cell basis. RESULTS: In serum-starved cultures, complete two-phase calcium signals and shorter, lower spikes occurred independent of cell cycle phase. However, the confluence of cell culture seemed essential for inducing a complete response because cells in sparse cultures exhibited mostly short spikes with lower peaks or no transients at all. CONCLUSION: Because cell confluence, by virtue of cell-cell contacts, is assumed to be an important regulator of proliferation, one is tempted to speculate that in transformed cells the ability to produce stronger growth signals upon reaching confluence and facing contact inhibition could provide a proliferative advantage.     

In vitro evaluation of natural marine sponge collagen as a scaffold for bone tissue engineering

The selection of a suitable scaffold matrix is critical for cell-based bone tissue engineering. This study aimed to identify and characterize natural marine sponges as potential bioscaffolds for osteogenesis. Callyspongiidae marine sponge samples were collected from the Fremantle coast of Western Australia. The sponge structure was assessed using scanning electron microscopy (SEM) and Hematoxylin and eosin. Mouse primary osteoblasts were seeded onto the sponge scaffold and immunostained with F-actin to assess cell attachment and aggregation. Alkaline phosphatase expression, von Kossa staining and real-time PCR were performed to examine the osteogenic potential of sponge samples. SEM revealed that the sponge skeleton possessed a collagenous fibrous network consisting of interconnecting channels and a porous structure that support cellular adhesion, aggregation and growth. The average pore size of the sponge skeleton was measured 100 to 300 μm in diameter. F-actin staining demonstrated that osteoblasts were able to anchor onto the surface of collagen fibres. Alkaline phosphatase expression, a marker of early osteoblast differentiation, was evident at 7 days although expression decreased steadily with long term culture. Using von Kossa staining, mineralisation nodules were evident after 21 days. Gene expression of osteoblast markers, osteocalcin and osteopontin, was also observed at 7, 14 and 21 days of culture. Together, these results suggest that the natural marine sponge is promising as a new scaffold for use in bone tissue engineering.     

Effect of strain on human dermal fibroblasts in a three-dimensional collagen sponge

Our aim was to design a simple compression system and investigate the influence of mechanical stress on skin-like structures. Many mechanical compression studies have employed intricate culture systems, so the relationship between extracellular matrix material and the response of skin cells to mechanical stress remains unknown. Our approach uses only glass vials, 6-well plates and standard laboratory equipment. We examined the influence of mechanical stress on human skin fibroblasts embedded within a collagen sponge. The results show that mechanical compression increases MMP-1 and MMP-2 release by the cells into the the cell culture. Our results suggest that pressure on the skin may affect extracellular matrix degradation through some as yet unidentified pathways and that IL-6 mRNA expression may be involved in this effect. Using our approach, the effects of static mechanical stress on protein expression by cells in the culture medium and in sponges can be easily examined, and therefore this system will be useful for further analyses of skin responses to mechanical stress.     

Cardiac differentiation at an initial low density of human-induced pluripotent stem cells

A high density of human-induced pluripotent stem cells (hiPSCs) improves the efficiency of cardiac differentiation, suggesting the existence of indispensable cell-cell interaction signals. The complexity of interactions among cells at high density hinders the understanding of the roles of cell signals. In this study, we determined the minimum cell density that can initiate differentiation to facilitate cell-cell interaction studies. First, we co-induced cardiac differentiation in the presence of the glycogen synthase kinase-3β inhibitor CHIR99021 and activin A at various cell densities. At an initial low density, cells died within a few days in RPMI-based medium. We then investigated the culture conditions required to maintain cell viability. We used a basal medium excluding important components for the maintenance of hiPSC pluripotency, including activin A, basic fibroblast growth factor, and insulin. Supplementation of the basal medium with Rho-associated protein kinase inhibitor and insulin improved cell viability. Interestingly, addition of basic fibroblast growth factor enabled the expression of cardiac markers at the mRNA level but not the protein level. After further modification of the culture conditions, 10% of the cells expressed the cardiac troponin T protein, which is associated with cell contraction. The novel protocol for cardiac differentiation at an initial low cell density can also be used to evaluate high cell density conditions. The findings will facilitate the identification of cell signals required for cardiomyocyte formation.     

Mariculture and Natural Production of the Antitumoural (+)-Discodermolide by the Caribbean Marine Sponge Discodermia dissoluta

Biotechnological research on marine organisms, such as ex situ or in situ aquaculture and in vitro cell culture, is being conducted to produce bioactive metabolites for biomedical and industrial uses. The Caribbean marine sponge Discodermia dissoluta is the source of (+)-discodermolide, a potent antitumoural polyketide that has reached clinical trials. This sponge usually lives at depths greater than 30 m, but at Santa Marta (Colombia) there is a shallower population, which has made it logistically possible to investigate for the first time, on ways to supply discodermolide. We thus performed in situ, 6-month fragment culture trials to assess the performance of this sponge in terms of growth and additional discodermolide production and studied possible factors that influence the variability of discodermolide concentrations in the wild. Sponge fragments cultured in soft mesh bags suspended from horizontal lines showed high survivorship (93 %), moderate growth (28 % increase in volume) and an overall rise (33 %) in the discodermolide concentration, equivalent to average additional production of 8 μg of compound per millilitre of sponge. The concentration of discodermolide in wild sponges ranged from 8 to 40 μg mL^?1. Locality was the only factor related to discodermolide variation in the wild, and there were greater concentrations in peripheral vs. basal portions of the sponge, and in clean vs. fouled individuals. As natural growth and regeneration rates can be higher than culture growth rates, there is room for improving techniques to sustainably produce discodermolide.     

Cultivation of Marine Sponges

There is increasing interest in biotechnological production of marine sponge biomass owing to the discovery of many commercially important secondary metabolites in this group of animals. In this article, different approaches to producing sponge biomass are reviewed, and several factors that possibly influence culture success are evaluated. In situ sponge aquacultures, based on old methods for producing commercial bath sponges, are still the easiest and least expensive way to obtain sponge biomass in bulk. However, success of cultivation with this method strongly depends on the unpredictable and often suboptimal natural environment. Hence, a better-defined production system would be desirable. Some progress has been made with culturing sponges in semicontrolled systems, but these still use unfiltered natural seawater. Cultivation of sponges under completely controlled conditions has remained a problem. When designing an in vitro cultivation method, it is important to determine both qualitatively and quantitatively the nutritional demands of the species that is to be cultured. An adequate supply of food seems to be the key to successful sponge culture. Recently, some progress has been made with sponge cell cultures. The advantage of cell cultures is that they are completely controlled and can easily be manipulated for optimal production of the target metabolites. However, this technique is still in its infancy: a continuous cell line has yet to be established. Axenic cultures of sponge aggregates (primmorphs) may provide an alternative to cell culture. Some sponge metabolites are, in fact, produced by endosymbiotic bacteria or algae that live in the sponge tissue. Only a few of these endosymbionts have been cultivated so far. The biotechnology for the production of sponge metabolites needs further development. Research efforts should be continued to enable commercial exploitation of this valuable natural resource in the near future. Received November 5, 1998; accepted June 20, 1999.     

Effect of hypertonic medium on human cell growth: III. Changes in cell kinetics of EUE cells

The effects of hypertonicity on cell kinetics of EUE cells in culture have been investigated. After 4 days of growth in a hypertonic medium, the plating efficiency of EUE cells was reduced and cell growth was significantly slowed. Flow cytometric measurements of DNA content in synchronized cells, as well as flow cytometric determinations of DNA content and bromodeoxyuridine incorporation in asynchronous cells, also showed that the cell cycle is slowed in a hypertonic medium. In addition, the fraction of cycling cells is smaller and their progression through the S phase slower than in an isotonic medium.     

In vitro development of Cryptosporidium parvum in serum-free media

AIM: To compare commercially available serum-free media with common, standard, growth medium for their ability to support growth of Cryptosporidium parvum in HCT-8 cell cultures. METHODS AND RESULTS: Twelve serum-free media formulations with or without additional supplements were tested against a standard growth medium containing 2% FBS in HCT-8 cell cultures. After a 48-h incubation period, the level of parasite development was determined by ELISA. The extent of development in the serum-free media was determined as a percentage of infections compared with those obtained using a standard growth medium. CONCLUSIONS: Several of the serum-free media formulations, which included MDCK, UltraMDCK, PC-1, UltraCHO and UltraCulture, compared favourably with a traditional, standard growth medium. Moreover, increasing FBS concentrations to 10% actually resulted in an overall decrease in development in many cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Several serum-free medium formulations are available which allow development of C. parvumin vitro at levels comparable with standard media employing FBS. These serum-free media are particularly useful for applications, which may require a more defined medium without the presence of FBS. Moreover, the elimination of FBS as a variable allows investigators the ability to more closely regulate their experimental systems when growing C. parvum in cell cultures.