Nutritional requirements of Plasmodium falciparum in culture. I. Exogenously supplied dialyzable components necessary for continuous growth

Continuous cultivation of Plasmodium falciparum presently requires the nutritionally complex medium, RPMI 1640. A basal medium of KCl, NaCl, Na2HPO4, Ca(NO3)2, MgSO4, glucose, reduced glutathione, HEPES buffer, hypoxanthine, phenol red (in RPMI 1640 concentrations), and 10% (v/v) exhaustively dialyzed pooled human serum was used to determine which vitamins and amino acids had to be exogenously supplied for continuous cultivation. Supplementation of basal medium with calcium pantothenate, cystine, glutamate, glutamine, isoleucine, methionine, proline, and tyrosine was necessary for continuous growth. This semi-defined minimal medium supported continuous growth of four isolates of P. falciparum at rates slightly less than those obtained with RPMI 1640. Adding any other vitamin or amino acid did not improve growth. Incorporation of several non-essential amino acids, particularly phenylalanine and leucine, into proteins was markedly enhanced in the minimal medium compared to RPMI 1640.     

Encystment and metacercariae development of Echinostoma cinetorchis cercariae in an in vitro culture system

For some species of 37-collar-spined Echinostoma, their cercariae successfully encyst and develop to metacercariae in vitro. In our study, we cultured Echinostoma cinetorchis cercariae in 12 different media to study the formation of metacercariae. Locke's solution, medium 199, and RPMI 1640 were used as media for culture. RPMI 1640 produced the highest encystment and normal metacercariae development. The osmolality of the media was related to their ability to encyst and develop. The 0.5 X media induced higher encystment and normal metacercaria formation than the 1x media. The addition of fetal bovine serum to RPMI 1640 increased the level of encystment and normal metacercariae development. In the mixture of 0.5 x RPMI 1640 and 10% fetal bovine serum, encystment was highest, at 96.0%, and the development ratio of normal metacercariae was also the highest, at 91.5%, after 48 hr of cultivation. In a viability test, 7 day- and 14 day-cultured metacercariae were successfully matured to adult worms in experimentally infected rats. These results showed that E. cinetorchis cercariae could be cultured to viable metacercariae in an in vitro culture system and that 0.5 x RPMI 1640 plus 10% fetal bovine serum was the most useful medium for cultivation. This culture system can be adapted for additional studies on the E. cinetorchis life cycle, especially to supply large numbers of metacercariae for other studies on this echinostome.     

Nutritional Requirements of Plasmodium falciparum in Culture. I. Exogenously Supplied Dialyzable Components Necessary for Continuous Growth

Continuous cultivation of Plasmodium falciparum presently requires the nutritionally complex medium, RPMI 1640. A basal medium of KCl, NaCl, Na₂HPO₄, Ca(NO₃)₂, MgSO₄, glucose, reduced glutathione, HEPES buffer, hypoxanthine, phenol red (in RPMI 1640 concentrations), and 10% (v/v) exhaustively dialyzed pooled human serum was used to determine which vitamins and amino acids had to be exogenously supplied for continuous cultivation. Supplementation of basal medium with calcium pantothenate, cystine, glutamate, glutamine, isoleucine, methionine, proline, and tyrosine was necessary for continuous growth. This semi-defined minimal medium supported continuous growth of four isolates of P. falciparum at rates slightly less than those obtained with RPMI 1640. Adding any other vitamin or amino acid did not improve growth. Incorporation of several non-essential amino acids, particularly phenylalanine and leucine, into proteins was markedly enhanced in the minimal medium compared to RPMI 1640.     

Induction of gametocytogenesis in Plasmodium falciparum by the culture supernatant of hybridoma cells producing anti-P. falciparum antibody

There have been many unsuccessful attempts to induce gametocytogenesis in vitro. In the present experiment, however, we found that RPMI-CS medium and RPMI-FS medium prepared by dissolving powdered RPMI 1640 medium in the culture supernatants of hybridoma cells, hybrid line D21 and 219.5, respectively, that produce anti-P. falciparum antibody induced gametocytogenesis. Gametocytogenesis was consistently observed from 3 days after addition of these media. The culture supernatant of anti-P. falciparum antibody producing hybridoma cells did not induce gametocytogenesis in the absence of RPMI 1640 medium. RPMI-MS medium, prepared by dissolving powdered RPMI 1640 medium in the culture supernatant of myeloma cells, SP2/O-Ag 14, which was used as a control, induced a few gametocytes.     

Increase of the antimicrobial susceptibility ofYersinia enterocolitica associated with the expression of the virulence plasmid

Virulent strains ofYersinia enterocolitica incubated in RPMI 1640 medium with 25 mM HEPES at 37°C were more susceptible to several antibiotics than their plasmid-free isogenic derivatives. The enumeration of viable bacteria in RPMI 1640 agar at 37°C to discriminate between plasmid-bearing and spontaneously derived, plasmid-free bacteria made it possible to show that the plasmid presence was associated with a fourfold decrease of minimal inhibitory concentration of ampicillin, streptomycin, gentamicin, chloramphenicol, and oxytetracycline. SinceY. enterocolitica is an intracellular pathogen and RPMI 1640 medium mimics the intracellular milieu, the plasmid-associated increase of susceptibility to antibiotics that are concentrated by animal cells may be of clinical relevance.     

Effect of media composition on the induction of chorionic gonadotropin by sodium butyrate in HeLa cells

Summary Production of the glycoprotein hormone α-subunit by HeLa cells and its induction by sodium butyrate are dependent on the choice of culture medium. Under identical growth conditions it was found that subunit synthesis in the presence of butyrate was highest in RPMI 1640, lowest in Medium 199 (M199), and intermediate in minimum essential medium (MEM) and Waymouth's MB 752/1. Cell growth was similar in all media examined and was retarded in the presence of butyrate. Alkaline phosphatase activity was also lower in M199 than in RPMI 1640, although, in general, the magnitude of this difference was less than that for the hormone subunit. Incorporation of [1-¹⁴C]butyrate by HeLa cells was simimar in both M199 and RPMI 1640, indicating that uptake and metabolism of the fatty acid were not significantly different under these conditions. In the presence of 3 mM butyrate, mixtures of RPMI 1640 and M199 gave intermediate levels of α-subunit and alkaline phosphatase compared to each medium alone. Intracellular levels of α-subunit as well as that of the culture medium were reduced in M199 compared to RPMI 1640 indicating that synthesis rather than secretion was altered. This work was supported by Grant CA 21534 from the National Institutes of Health, Bethesda, MD.     

Simultaneous identification of T and B cells in blood smears using antibody coated bacteria.

Rabbit lymph nodes produce in vitro more antibodies over longer periods if the culture medium is supplemented with a mixture of horse and fetal bovine serum rather than rabbit serum. Medium RPMI 1640 sustains longer antibody responses and promotes a greater expansion of the cultures than medium 199, both media being supplemented with the serum mixture. Propagation of fast-growing, antibody-producing cultures fed with RPMI 1640 was accomplished by interculture transfer of lymphoid tissue. Round, mostly nonadherent cells grow in RPMI 1640 and medium 199 but in the former a striking proliferation of fibroblast-like cells takes place. This proliferation does not occur in medium 199. Preliminary results indicate that although the fibroblast-like cells do not produce antibodies, they contribute to the response in vitro.     

Effects of DMEM and RPMI 1640 on the biological behavior of dog periosteum-derived cells

Periosteum-derived cells (PDCs) are being extensively studied as potential tissue engineering seed cells and have demonstrated tremendous promise to date. There is convincing evidence that culture medium could modulate the biological behavior of cultured cells. In this study, we investigate the effects of DMEM (low glucose) and RPMI 1640 on cell growth and cell differentiation of PDCs in vitro. PDCs isolated from Beagle dogs were maintained in DMEM and RPMI 1640, respectively. Then, the cell migration rate of periosteum tissues was analyzed. PDCs of the third passage were harvested for the study of proliferation and osteogenic activity. Proliferation was detected by MTT assay. Alkaline phosphatase activity and mineralized nodules were measured to investigate osteogenic differentiation. Our data demonstrated that DMEM induced alkaline phosphatase activity and strongly stimulated matrix mineralization in cell culture, while similar cell migration rates and proliferation behaviors were observed in the two culture conditions. Interestingly, the osteogenic differentiation of PDCs could be enhanced in DMEM compared with that in RPMI 1640. Thus, it can be ascertained that DMEM may serve as a suitable culture condition allowing osteogenic differentiation of dog PDCs.     

Relationship Between Susceptibility of <Emphasis Type="Italic">Candida</Emphasis> spp. Isolates to Amphotericin B and Death or Survival of Patients with Candidemia Episodes

In this study, the susceptibility to amphotericin B of Candida spp. isolates obtained from patients with candidemia was related to their respective clinical outcomes. The susceptibility tests were carried out in three culture media: RPMI 1640, Antibiotic medium 3 and Yeast Nitrogen Base dextrose. We have found that minimal inhibitory concentrations and minimal fungicidal concentrations obtained using AM3 and YNBd media were significantly higher for Candida spp. from patients who died than for those from patients who survived the candidemia (P < 0.05). The assays with RPMI 1640 medium did not show these differences.     

Inhibitory effect of extracellular histidine on cobalt-induced HIF-1alpha expression

Cobalt chloride (CoCl(2)) can mimic hypoxia in inducing hypoxia-inducible factor 1 (HIF-1). Several cultured cells were examined for susceptibility to CoCl(2) in DMEM, MEM and RPMI 1640 medium. Here we report that HIF-1α expression of mammalian cells by CoCl(2) was largely dependent on the culture medium. HIF-1α protein and hypoxia response element (HRE)-dependent reporter activity were strongly induced in RPMI 1640 but not in DMEM in several cultured cells including MCF-7, a human breast cancer cell line. Analysis of causal nutrients has revealed that histidine, which is contained richer in DMEM, acts as the inhibitory nutrient for cobalt-induced HIF-1α expression of MCF-7 cells in DMEM. D-Histidine also inhibited the HIF-1α activity at the same level as L-histidine, suggesting that sequestration of free cobaltous ion by chelation with histidine was the cause of the inhibition. These results demonstrate that selection of the culture medium must be considered with caution in cell culture experiments using CoCl(2) as a hypoxia-mimetic reagent.     

Generation of dendritic cells from human peripheral blood monocytes--comparison of different culture media

Culture medium or medium supplement is one of the factors responsible for dendritic cell (DC) generation, but little is known about the influence of various media on DC culture. In our study we generated DC from adherent monocytes of human peripheral blood in the presence of GM-CSF, IL-4 and TNF-alpha. The following culture media were used: RPMI 1640 supplemented with 2% human serum albumin; RPMI 1640 supplemented with 2% TCH serum replacement; X-VIVO 15 and Panserin 501. Flow cytometry analysis revealed that in all media cells were CD83+ and lost CD14. Interestingly, the use of Panserin and RPMI with albumin preferentially gave rise to CD1a+ DC, whereas in X-VIVO and RPMI with TCH we observed both CD1a+ and CD1a-. Our results showed that RPMI with TCH yielded the highest percentage of cells expressing both CD80 and CD86 molecules and, in contrast to other media, the higher percentage of CD86+ cells in comparison to CD80+ cells.     

Comparison of different methods for erythroid differentiation in the K562 cell line

Objective

To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis.

Results

Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001).

Conclusions

Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.

     

Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production

Existence of autocrine growth factors (aGFs) may influence the serum requirement for growth of hybridoma cells and thus significantly influence process economics. For the murine hybridoma cell line S3H5/2bA2, critical inoculum density (cID) and serum requirement for growth were inversely related for cultivation in both T flasks and spinner flasks. In spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 10³ cell/ml was necessary in RPMI 1640 medium with 10% serum. In T flasks, where the local cell density is higher than in spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 1 cell/ml was also necessary in RPMI 1640 medium with 10% serum. Further, immobilized cells at high local cell density could grow under conditions where cells in T flasks at corresponding overall cell density could not grow. The cells at high inoculum density were less sensitive to shear induced by mechanical agitation than the cells at low inoculum density. Taken together these observations support the existence of secreted aGF(s) by the hybridoma cell line used. Since the specific MAb production rate was independent of cultivation method and inoculum density, the existence of autocrine growth factors would suggest that the use of immobilized cells should improve the economics of MAb production.     

Evaluation of culture media for effects on cell cycle kinetics and incidence of chromosomal aberrations in human blood cells

Peripheral blood samples from 17 apparently healthy male volunteers were set up in duplicate cultures using three commercially available media: Eagle's MEM, RPMI 1640, and TC 199. BUdR (5-bromo,2-deoxyuridine) (10 micrograms/mL) was added to one of the cultures from each person in each medium after 24 h of culture initiation. All cultures were harvested at 72 h of incubation in the presence of colcemid. RPMI 1640 stimulated the highest mitotic activity in both BUdR-treated and untreated cultures. Higher numbers of first division metaphases corresponded with the higher frequency of chromosome-type aberrations in cultures with Eagle's MEM as compared with RPMI 1640 media. On the other hand, higher numbers of chromatid-type aberrations were present in cultures with TC 199 as compared with those with Eagle's MEM. When the chromosome- and chromatid-type aberration data were pooled to score total cytogenetic abnormalities, an influence of the medium was demonstrable. While cultures with Eagle's MEM and TC 199 had the greater number of first division cells, third of subsequent division cells were most prevalent in RPMI 1640 cultures. It is inferred that the length of the cell cycle, the mitotic index, and to some degree the incidence of spontaneous cytogenetic abnormalities are variable attributes of culture media.     

Optimisation of media for the culture of normal human epithelial cells from oesophageal mucosa

A technique has been developed which allows growth and histology/cytochemistry of primary oesophageal mucosal explant cultures to be monitored over four weeks. The paper describes experiments designed to optimise media and culture conditions. The results suggest that optimal growth can be obtained in media containing RPMI 1640, and 10% horse or newborn calf serum. McCoy's 5A medium or Dulbecco's modified MEM could be substituted for RPMI but Iscove's serum-free medium or Ham's nutrient mixture inhibited growth or promoted fibroblast contamination. The essential additive appeared to be insulin while selenium was highly toxic to the cells. Hydrocortisone or EGF improved growth slightly under some conditions. Neither transferrin nor cholera toxin had any beneficial effect. None of the cell culture flask coating agents improved attachment or growth.     

Long-term in vitro cultivation of Plasmodium berghei

Long-term in vitro culture of Plasmodium berghei was established using the Petri dish candle jar method of Trager and Jensen (1976). Cultures were established at 22, 27 and 37°C. As optimal growth was observed at 27°C, subsequent cultivation was carried out at this temperature. RPMI 1640 medium was modified by incorporating additional glucose (1 mg ml⁻¹) and bactopeptone (1 mg ml⁻¹) in the medium. This medium was found suitable for maintenance of mouse erythrocytes in vitro. P. berghei cultures were maintained using candle jars and this modified RPMI 1640 medium for 45 weeks.     

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10⁶ cells ml^–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10⁵ cells ml^–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10⁶ cells ml^–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum     

Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes.

fms genes encoding either wild-type or constitutively activated colony-stimulating factor 1 receptors (CSF-1R) were introduced by retroviral infection into long-term mouse lymphoid cultures. Four early pre-B-cell lines transformed by the feline v-fms oncogene underwent spontaneous and irreversible differentiation to macrophages when transferred from RPMI 1640 to Iscove modified Dulbecco medium. Expression of wild-type human CSF-1R in early pre-B cells conferred no proliferative advantage unless human CSF-1 was added to the culture medium. A clonal, factor-dependent early pre-B-cell line (D1F9), selected for continuous growth on NIH 3T3 cell feeder layers producing human CSF-1, could be maintained in RPMI 1640 medium containing interleukin-7 (IL-7) but also differentiated to macrophages when grown in Iscove modified Dulbecco medium containing human CSF-1. The macrophages retained parental immunoglobulin gene rearrangements and proviral insertions, lost B-cell antigens, expressed butyrate esterase and MAC-1, were actively phagocytic, and no longer survived in IL-7. Unlike factor-independent v-fms transformants, the irreversible commitment of D1F9 cells to differentiate in the macrophage lineage could be suppressed by IL-7, depended on human (but not mouse) CSF-1, and was inhibited by an antibody to human CSF-1R. Signals mediated by transduced CSF-1R can therefore play a deterministic role in cell differentiation.     

Relative ranking of culture media based on proliferation kinetics and spontaneous chromosomal aberrations in PHA-responsive human peripheral blood lymphocytes.

Proliferation kinetics and spontaneous yield of chromosomal aberrations phytohemagglutinin (PHA)-responsive peripheral blood lymphocytes were studied from blood samples collected from 45 individuals in 4 different synthetic media. Except for a significant difference for Eagle's MEM and RPMI 1640, the other media did not show difference for the yield of chromatid or chromosome type of aberrations. Differences were however noticed in the proliferation kinetics (mitotic and proliferative rate indices) of cells among the media used. The study indicated that (i) the intrinsic properties of media which influence proliferation rate and yield of chromosomal aberrations are independent of each other as higher proportion of first division cells do not correspond with higher frequency of chromosomal aberrations, (ii) the amount of free-radical scavengers present in the medium, apart from the genetic make-up of the individuals, may contribute to the spontaneous yield of chromosomal aberrations and (iii) RPMI 1640 medium, which showed higher transformation and faster cycling rate for the lymphocytes, may be considered as medium of choice for analysing two main cytogenetic end-points, chromosomal aberrations and sister chromatid exchanges (SCEs).     

Self-propagating calciferous particles detected in a human cell line Kasumi-6 (JCRB 1024)

Summary Tiny particles were found in the medium in the presence of the human leukemia cell line Kasumi-6. The particles were separated from human cells by filtration and incubated in RPMI1640 supplemented with 10% fetal calf serum at 37 C. The particles increased in number very slowly in the liquid medium but did not reveal any biological activity. Transmission electron microscopy of the particles showed a spheroid or ovoid shape in ultrathin section. No specific polypeptides from the purified particles were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), except for bovine fetuin that adsorbed to the surface of the particles. X-ray diffractometry as well as Fourier transform infrared spectrometry suggested the particles consisted of hydroxyapatite. The mechanism of self-propagation of the hydroxyapatite particles in liquid medium is currently unknown. This type of particle has been overlooked for a long period because it is noncultivable. It will be necessary to examine its biological effects to the cultured cells.