In vitro cultivation of Plasmodium falciparum: studies with modified medium supplemented with ALBUMAX II and various animal sera

RPNI, a combination of three commercially available growth media (RPMI-1640, NCTC-135 and IMDM) has been found to support long term continuous cultivation of 3D7 strain of Plasmodium falciparum in the presence of 10% bovine calf serum. During the present study, the suitability of this medium was evaluated for the development of P. falciparum in the presence of horse, goat and rabbit sera as well as various concentrations of ALBUMAX II. RPNI medium supplemented with 10% bovine calf serum (RPNI-BCS) was used as control. The cultures were maintained in candle jars protocol and parasitaemia was monitored daily up to day 7. Horse, goat and rabbit sera all supported the development of P. falciparum. Horse serum gave best results in RPNI medium and supported continuous culture up to day 100. The parasitaemia in the presence of ALBUMAX was significantly higher in RPNI than in RPMI-1640. Addition of hypoxanthine in RPMI-1640 caused an increase in parasitaemia whereas no obvious advantage could be observed in RPNI. The findings exhibited that medium RPNI has an edge over conventional RPMI-1640 medium for in vitro cultivation of P. falciparum.     

Effect of Arginine-deficient Media on the Herpes-Type Virus Associated with Cultured Burkitt Tumor Cells

After incubation at 37 C for 2 or more days, Basal Medium Eagle (BME) supplemented with 25% fetal calf serum (FCS) was not able to support adequate growth of EB3 and other lines of Burkitt tumor cells. The medium did, however, increase, by a factor of about 10, the number of cells synthesizing herpes-type virus with which the cultures were persistently infected. Not every lot of FCS produced these effects, nor were these effects seen when BME and FCS were incubated separately for 7 days before the medium was completed. At 37 C, appropriate lots of FCS interacted with several of the amino acids present in BME; this interaction resulted in an inhibition of cellular growth, whereas interaction with arginine yielded the virus-enhancing effect. Arginine-free BME, supplemented with 25% FCS and used without prior incubation, prevented cellular replication and promoted viral infection to a similar extent as did preincubated complete medium. Replenishment of arginine reduced, but did not regularly abolish, the virus-enhancing activity of preincubated media. RPMI-1629 medium was less affected by preincubation with FCS because it contained twice the amount of arginine that BME contained. The FCS factors which act upon arginine and other amino acids are not dialyzable and are partially resistant to heating at 56 or 60 C for 30 to 60 min. Calf and horse sera appear to be devoid of these activities. The nature of these interactions, as well as the mechanism by which arginine deficiency enhanced the viral infection, remains to be ascertained.     

In Vitro Maintenance of Clonorchis sinensis Adult Worms

Clonorchis sinensis is a biological carcinogen inducing human cholangiocarcinoma, and clonorchiasis is one of the important endemic infectious diseases in East Asia. The present study investigated survival longevity of C. sinensis adult worms in various in vitro conditions to find the best way of keeping the worms longer. The worms were maintained in 0.85% NaCl, 1×PBS, 1×Locke''s solution, RPMI-1640, DMEM, and IMDM media, and in 1×Locke''s solution with different supplements. All of the worms died within 3 and 7 days in 0.85% NaCl and 1×PBS, respectively, but survived up to 57 days in 1×Locke''s solution. The worms lived for 106 days in DMEM, and 114 days in both RPMI-1640 and IMDM media. The survival rate in RPMI-1640 medium was the highest (50%) compared to that in DMEM (20±10%) and in IMDM (33.3±25.2%) after 3 months. The 1×Locke''s solution with 0.005% bovine bile supplement showed increased duration of maximum survival from 42 days to 70 days. Higher concentration of bile supplements than 0.005% or addition of glucose were disadvantageous for the worm survival. The worms died rapidly in solutions containing L-aspartic acid, L-glutamic acid, and adenine compared to L-arginine, L-serine, and L-tryptophan. In conclusion, the 1×Locke''s solution best supports the worms alive among inorganic solutions for 57 days, and the RPMI-1640 medium maintains living C. sinensis adults better and longer up to 114 days in vitro than other media.     

Culture of in vitro fertilized rabbit ova

Embryonic development of in-vitro fertilized rabbit ova was assessed following in-vitro culture in four different serum supplemented media. A mixture of Basal Medium Eagle (BME) and Ham's F10 medium (1:1) provided better support for in-vitro development than Ham's F10, BME, or regular acidic saline (RAS). In-vitro embryonic development in the BME/Ham's F10 mixture was synchronous with in-vivo development through at least 55 hr of culture. After 54 hr of culture, embryos transferred to the oviduct of a synchronous pseudopregnant recipient were able to implant at the same rate as simultaneously transferred embryos grown in vivo. BME/Ham's F10 supplemented with 10% newborn calf serum was highly supportive of rabbit embryo development following in-vitro fertilization.     

Improved Chemically Defined Basal Medium (CMRL-1969) for Primary Monkey Kidney and Human Diploid Cells

An improved tissue culture basal medium, CMRL-1969, supplemented with serum, has been evaluated by measuring the growth responses of primary cultures of trypsin-dispersed monkey kidney cells (PMKC) and of an established culture of a human diploid cell strain (HDCS). Medium H597, an early modification of medium 199 which has been used successfully in the preparation of poliomyelitis vaccine for 15 years, was used for comparison. In addition, parallel testing was done with Basal Medium Eagle (BME) widely used for the growth of HDCS. The improvements in basal medium CMRL-1969 are attributed to changes in amino acid concentrations, in vitamin composition, and, in particular, to enhanced buffering capacity. The latter has been achieved by the use of free-base amino acids and by increasing the dibasic sodium phosphate. The new medium has already been used to advantage for the production of polioviruses in PMKC where equivalent titers were obtained from cultures initiated with 70% of the number of cells required with earlier media. The population-doubling time was reduced in this system. Also, with small inocula of HDCS, the time required to obtain maximum cell yield was shorter with CMRL-1969 than with BME. Both media were supplemented with 10% calf serum. Maximum cell yields after repeated subcultivation in the new basal medium were greatly increased and the stability of the strain, as shown by chromosomal analysis, was not affected. Basal medium CMRL-1969 can be prepared easily in liquid or powdered form.     

Innovative serum-free medium for in vitro cultivation of promastigote forms of Leishmania species

We described a comparatively simple medium formula (CML) using common, available and reasonably priced ingredients that could be used in place of medium that requires calf serum enhancement for cultivation of Leishmania promastigote forms. This medium equivalently supported the growth of parasites at rates comparable with those obtained with serum supplemented RPMI-1640 medium. Leishmania promastigotes reproduced in CML exhibited moderate to high infectivity capacities when tested against J774 macrophage cell line. No significant difference was noted between Leishmania strains cultivated in the newly modified medium and those grown in RPMI-1640 medium in their cells infectivity and replication potentials. The use of new CML can easily take the place of other biphasic or liquid media because of its easy preparation and instantaneous use, reasonable price, availability of ingredients, and its long shelf life, which is 30–45 days. The fact that this medium is similar to other culture media as far as durability and quantity of produced parasites might give it an advantage over the other currently used media.     

Glucagon production by cultured pancreatic islets: effects of different culture conditions and media

Various conditions for tissue culture of collagenase-isolated mouse pancreatic islets were studied for their effects on the glucagon production of the cultured specimens. Culture media containing heat-treated bovine calf serum degraded [125I]glucagon to a much less extent than those supplemented with untreated serum. Addition of aprotinin to the heat-treated serum gave a further reduction of the [125I]glucagon degradation in the culture medium. A similar supplementation of Medium 199, used for culture of isolated islets, resulted in the most extensive glucagon accumulation in the culture medium. Islets cultured free-floating or attached to the bottom of the culture dishes contained similar amounts of glucagon. However, the free-floating islets released less glucagon when tested in short-term experiments performed at the end of the 1 wk culture period. A comparison between different culture media showed that islets cultured in RPMI-1640 had the highest glucagon content and released most glucagon to the culture medium. Moreover, these islets responded most actively to an acute arginine challenge at the end of the culture period. The present data suggest that the optimal conditions for culture of isolated islets aimed at studies of glucagon production may be obtained by using a culture medium consisting of RPMI-1640 supplemented with both a proteinase inhibitor and heat-inactivated serum.     

Effect of substrata and fibroblast growth factor on the proliferation in vitro of bovine aortic endothelial cells

The hypothesis that, in the case of clonal or low-density cultures, cells which do not readily proliferate are those that do not produce an extracellular matrix (ECM), while those that proliferate actively are cells that have retained their ability to produce it, has been tested using low-density vascular endothelial cell cultures maintained on either plastic or ECM-coated dishes and exposed to various combinations of media and sera. Proliferation of low-density vascular endothelial cell cultures seeded on plastic and exposed to DMEM, RPMI-1640, or medium 199 plus thymidine is a function of the batch of calf serum used to supplement the various media. In all three cases, such cultures proliferated at a slow rate and fibroblast growth factor (FGF) greatly accelerated their proliferation. In contrast, when similar cultures were seeded on ECM-coated dishes, they actively proliferated regardless of the batch of calf serum to which they were exposed. FGF was no longer required in order for cultures to become confluent. In the case of cultures exposed to RPMI-1640 or medium 199 plus thymidine, it was even toxic. When cultures were exposed to either medium 199 or Waymouth medium, cells did not proliferate, regardless of the substrate (either plastic or ECM) upon which they were maintained and of the batch of serum to which they were exposed. Addition of FGF to such media had no effect. It is therefore likely that nutrient limitations in both of these media restrict the ability of low-density vascular endothelial cells to respond to the mitogenic stimuli provided by either serum or FGF. These restrictions cannot be relieved by maintaining cells on ECM-coated dishes, and modifications of the nutrient composition of both media is required in order to allow cells to respond to either FGF or serum when maintained on plastic or to serum alone when maintained on ECM. These results suggest that, when low-density cell cultures are maintained on plastic and exposed to an adequate medium, their proliferation will be a function of both serum and FGF. When maintained on ECM, their proliferation will depend only on serum. It is therefore possible that the inability of serum to stimulate optimal cell proliferation when cells are maintained on plastic results from an inability of the cells to produce an ECM, and that FGF could induce such production.     

Glucagon production by cultured pancreatic islets: Effects of different culture conditions and media

Summary Various conditions for tissue culture of collagenase-isolated mouse pancreatic islets were studied for their effects on the glucagon production of the cultured specimens. Culture media containing heat-treated bovine calf serum degraded [¹²⁵I]glucagon to a much less extent than those supplemented with untreated serum. Addition of aprotinin to the heattreated serum gave a further reduction of the [¹²⁵I]glucagon degradation in the culture medium. A similar supplementation of Medium 199, used for culture of isolated islets, resulted in the most extensive glucagon accumulation in the culture medium. Islets cultured free-floating or attached to the bottom of the culture dishes contained similar amounts of glucagon. However, the free-floating islets released less glucagon when tested in short-term experiments performed at the end of the 1 wk culture period. A comparison between different culture media showed that islets cultured in RPMI-1640 had the highest glucagon content and released most glucagon to the culture medium. Moreover, these islets responded most actively to an acute arginine challenge at the end of the culture period. The present data suggest that the optimal conditions for culture of isolated islets aimed at studies of glucagon production may be obtained by using a culture medium consisting of RPMI-1640 supplemented with both a proteinase inhibitor and heat-inactivated serum. This work was supported by grants from the Swedish Medical Research Council (12X-109), the Nordic Insulin Fund, and the Swedish Diabetes Association.     

A comparison of different nutrient media and supplementation with dexamethasone for mouse colon organ culture

Several complex nutrient media were compared for their effectiveness in maintaining viable and functional mouse colon mucosa in organ culture. The order of superiority for preserving survival of normal tissues for 14 days was: Williams' Medium E > Morgan's 199 > CMRL-1066 > Waymouth's MB 752/1 > Eagle's MEM > Trowell's T8. The ₃H-thymidine labeling index was highest in colon explants maintained in Morgan's 199 > Williams' Medium E > Waymouth's MB 752/1 > CMRL-1066 > Eagle's MEM. However, the very high labeling produced by Morgan's 199 medium was abnormal in comparison to in vivo levels. Supplementation with 1.0 µM dexamethasone almost always improved crypt survival and maintained normal DNA synthetic activity.Supported by National Cancer Institute contract No. 1-CP-75952 and grant No. CA-29602.     

In Vitro Regression of Tadpole Tail by Thyroid Hormone

DERBY successfully maintained the tail of tadpole ( Rana pipiens) in vitro over a period of 2 weeks in a physiological salt solution (1). When we tried to apply DERBY'S methods of the tissue culture to tadpoles of bullfrog, Rana catesbeiana , it was found that the tissue regressed spontanously without stimulation of thyroid hormone. Several different media were examined in order to select a better culture medium for the bullfrog tadpole tissues. RPMI-1640 medium supplemented with insulin and transferrin was found to be satisfactory for this aim. With this improved medium, the interaction between the epidermis and the mesenchyme has been investigated during the hormone-induced tadpole tail regression and the epidermal dependence of the mesenchyme regression was demonstrated by the following three experiments. (i) Some of surgically prepared mesenchymes regressed in responce to thyroid hormone. In these cases the mesenchymes were revealed to be contaminated with the remaining epidermal cells. (ii) Complete removal of the epidermis was accomplished by the chemical treatment. The mesenchyme thus obtained ("nude tail fin") was insensible to thyroid hormone. (iii) "Skin conditioned medium" (SCM) was prepared by culturing the skin in the presence and absence of thyroid hormone. Nude tail fin regressed when cultured in the SCM containing thyroid hormone.     

Effects of culture media on spontaneous incidence of mitotic index, chromosomal aberrations, micronucleus counts, sister chromatid exchanges and cell cycle kinetics in peripheral blood lymphocytes of male and female donors

The spontaneous incidence of mitotic index (MI), chromosomal aberrations (CA), micronucleus counts (MNC), sister chromatid exchanges (SCE) and cell cycle kinetics (CCK) were studied in human peripheral blood lymphocytes grown in M199 and RPMI-1640 culture media. Lower frequencies of CAs, MNC and SCEs were observed in lymphocytes cultured in medium RPMI-1640. The reduction of the MI and the replicative index in M199 medium showed delayed cell cycle kinetics.     

Parasitic pattern of rodent Plasmodium in blood from mouse tail and from Anopheles blood meal

During the course of the infection of mice by rodent malaria parasites (P. yoelii yoelii, P. vinckei petteri, and P. chabaudi chabaudi) the parasitic pattern in the blood smears differed according to the site of sampling: mosquito blood meal (usually actively sucked from capillaries) or mouse tail (issued from larger vessels). There were fewer old trophozoites and mature schizonts, and more immature schizonts, in the mosquito blood meal than in the tail blood of mice. It is suggested that the erythrocyte containing a schizont becomes less rigid when merozoites formation begins. The greater abundance in the mosquito blood meal of the gametocytes infective stage (0-I for P. yoelii, II for P. vinckei), previously shown, could be due to a particular flexibility of this stage, a character progressively selected.     

Enhancement of phenotypic expression in cultured malignant liver epithelial cells by a complex medium.

The ability of two media identically supplemented with serum to maintain functional and structural differential properties of cultured malignant liver epithelial cells was compared. Reuber H35 hepatoma cells grown in a complex medium, Medium E, possessed the gluconeogenic enzyme phosphoenolpyruvate carboxykinase and bile canaliculi. When grown in Eagle's Minimal Essential Medium, the activity of phosphoenolpyruvate carboxykinase was reduced and bile canaliculi were not formed. This decrease in properties was restored when cells maintained in Minimal Essential Medium was returned to Medium E. It was concluded that the enhanced phenotypic expression was probably due to enrichment of the nutrient environment.     

Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, protein-free culture medium

In Exp. 1, Medium 199 and Medium RD (RPMI-1640 and Dulbecco's MEM, 1:1 v/v) were compared in a 2 x 2 factorial design by supplementing each with 15 mg bovine serum albumin (BSA)/ml of 1 mg polyvinyl alcohol (PVA)/ml. All media contained 5 micrograms insulin/ml, 5 micrograms transferrin/ml, 5 ng selenium/ml (ITS), and 10 ng epidermal growth factor (EGF)/ml. One-cell embryos were cultured at 39 degrees C with 5% CO2 in air for 65 h and then stained with Hoechst 33342 to determine blastomere number. Embryos in Medium 199 developed poorly (P less than 0.001) when PVA was used instead of BSA (30 vs 76 cells/embryo), but developed rapidly in Medium RD with PVA or BSA (118 and 121 cells). Similar results were obtained in Exp. 2 in BSA- and PVA-free medium. In Exp. 3, the development of 1-cell embryos after 65 h in unsupplemented (protein-free) Medium RD (68% blastocysts, 117 cells) did not differ (P greater than 0.37) from that obtained using Medium RD with insulin, ITS or EGF alone. Culture in protein-free Medium RD for 96 h resulted in 82% of the 1-cell embryos forming blastocysts and 40% hatching through the zona pellucida. In a preliminary test of viability, 1-cell embryos cultured in this medium for 48 or 65 h and transferred to synchronous recipients resulted in 5/18 (28%) and 3/24 (12%) Day-15 viable fetuses. Cell counts of approximately 120 per blastocyst after culturing 1-cell embryos for 65 h in Medium RD indicated that cell division was more rapid than that obtained with all other media tested previously in this laboratory. This is the first report of rabbit embryo development from the 1-cell to the hatching blastocyst stage in a defined protein-free culture medium.     

Effector mechanism of host resistance in murine giardiasis: specific IgG and IgA cell-mediated toxicity

The role of specific serum and milk anti-Giardia muris antibodies in mediation of host-effector responses to this enteric pathogen is unknown. We have investigated antibody-dependent cell-parasite interactions, potentially important as mediators of protection against infection at the mucosal surface. Elicited mouse peritoneal neutrophils and macrophages were incubated with G. muris trophozoites in the presence of either serum or milk antibodies, and their adherence and phagocytosis of the parasites were assessed. The percentage of trophozoites with adherent neutrophils increased significantly in the presence of heat-inactivated immune rabbit serum (93.5% +/- 6.5) and immune mouse milk (54.4% +/- 11.3) and their purified IgG (35.2% +/- 9.7) and secretory IgA fractions (48.0% +/- 12.3) when compared with incubation in RPMI-10% FCS (21.7% +/- 13.9). Similarly, macrophage adherence to trophozoites increased from 49.7% +/- 14.3 in medium alone to respective values of 92.8% +/- 7.1 in immune rabbit serum and 77.3% +/- 11.0 in immune milk. Phagocytosis of parasites by macrophages also was enhanced after incubation in immune rabbit serum (48.0% +/- 4.0) and immune mouse milk (35.0% +/- 5.0) when compared with the percentage of trophozoites ingested when cells and parasites were incubated in RPMI-10% FCS (3.3% +/- 3.0). Transmission electron microscopy showed ingestion of parasites by neutrophils or macrophages after 15 min of incubation. Morphologic evidence of intracellular parasite injury was observed at 6 hr. A decrease in parasite infectivity also resulted when trophozoites were incubated with neutrophils or macrophages and a source of antibodies, and intragastrically fed to weanling mice. These observations show that both antitrophozoite IgG, secretory IgA, and mouse phagocytic cells interact in vitro to promote parasite clearance. Because both the humoral and cellular components of this system are found intraluminally in the small intestine and in milk, they may represent a biologically relevant protective response against giardiasis.     

Formation of a close junction during invasion of erythrocytes by Toxoplasma gondii in vitro

Michel R., Schupp K., Raether W. and Bierther F.W. 1980. Formation of a close junction during invasion of erythrocytes by Toxoplasma gondii in vitro. International Journal for Parasitology10: 309–313. During the investigation of erythrocyte entry by Toxoplasma trophozoites the formation of a close junction between the erythrocyte and the invading parasite at the site of contact could be observed. The erythrocyte membrane and the outer membrane of the parasite pellicle show increased electron density in the circumferential attachment zone at the orifice of the erythrocyte invagination. The erythrocyte membrane has a thickness in this area of 7–8.5 nm. This membrane differentiation can be observed at the early stages of invasion when the tip of the apical end of the parasite induces a small erythrocyte invagination.It is still preserved in further advanced stages, when the parasite enters the enlarging invagination of the erythrocyte. As the morphological features resemble those observed during merozoite invasion into erythrocytes we consider our findings may have a similar significance for the mechanism of invasion by Toxoplasma as for the erythrocyte entry by malaria parasites.     

Effect of phosphatidylcholine-cholesterol liposomes on Entamoeba histolytica virulence

Trophozoites of Entamoeba histolytica HM-1:IMSS become less virulent after long-term maintenance in axenic cultures. The factors responsible for the loss of virulence during in vitro cultivation remain unclear. However, it is known that in vitro cultivation of amoeba in culture medium supplemented with cholesterol restores their virulence. In this study, we analyzed the effect of adding phosphatidylcholine-cholesterol (PC-Chol) liposomes to the culture medium and evaluated the effect of this lipid on various biochemical and biological functions of E. histolytica HM-1:IMSS in terms of its virulence. The addition of PC-Chol liposomes to the culture medium maintained the virulence of these parasites against hamster liver at the same level as the original virulent E. histolytica strain, even though these amoebae were maintained without passage through hamster liver for 18 months. The trophozoites also showed increased endocytosis, erythrophagocytosis, and carbohydrate residue expression on the amoebic surface. Protease activities were also modified by the presence of cholesterol in the culture medium. These findings indicate the capacity of cholesterol to preserve amoeba virulence and provide an alternative method for the maintenance of virulent E. histolytica trophozoites without the need for in vivo procedures.     

Antimicrobial Susceptibility Testing of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Determined by Microdilution Method Using a New Medium

Antibiotic susceptibility testing of Helicobacter pylori isolates was performed by broth microdilution method with MegaCell^TM RPMI-1640 Medium (SIGMA). Fifty five clinical isolates of H. pylori were tested against metronidazole, tinidazole, amoxicillin, and clarithromycin. The results were compared to those obtained by standard agar dilution method. The microdilution method performed with new medium, showed excellent correlation with agar dilution results, with 100% agreement for metronidazole, 96.3% for amoxicillin, 90.7% for clarithromycin, and 92.8% for tinidazole. MICs determined by proposed method were highly reproducible: replicate results were variable within one-two-fold dilution by using different inocula and different batches of medium.     

Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors

A serum free medium was developed, that could be used for the large scale propagation of various cell lines in bioreactors. The medium is based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's Medium F12, supplemented with transferrin, insulin and a BSA/oleic acid complex. Several myelomas, hybridomas derived from different myelomas and spleen cells, and other lymphoid and non-lymphoid cell lines were cultivated at growth rates comparable to those observed using serum-supplemented media. There was furthermore no reduction in the formation of products such as monoclonal antibodies or recombinant human interleukin-2.Abbreviations Ag8 Mouse myeloma cell line P3-X63-Ag8.653 - BME Basal Medium Eagle - BSA Bovine Serum Albumin - DMEM Dulbecco's Modified Eagle's Medium - EDTA Ethylenediaminete-traacetic Acid - e-PC Phosphatidyl choline from egg yolk - FCS Fetal Calf Serum - FGF Fibroblast Growth Factor - GHL Glycyl-histidyl-lysine - HDL High Density Lipoprotein - HPL Human Plasma Lipid - IF 1:1 mixture of IMDM and Ham's F12 - IMDM Iscove's Modified Dulbecco's medium - LDL Low Density Lipoprotein - NS1 Mouse myeloma cell line NSI-1-Ag4-1 - PBS Phosphate Buffered Saline - s-PC Phosphatidylcholine from soy beans - s-PE Phosphatidylethanolamine from soy beans - s-lecithin lecithin from soy beans