Propagation and Growth Cycle of Rickettsia quintana in a New Liquid Medium

The growth cycle of Rickettsia quintana was studied for the first time in liquid culture. Growth of the microorganism in a transparent broth medium was made possible by the finding that fetal calf serum (FCS), but not calf serum (CS), satisfied the requirement of R. quintana (Fuller strain) for red blood cell lysate. The three constituents of the medium, other than FCS, were autoclavable. The growth cycle was characterized by a lag phase of approximately 24 hr, an exponential growth phase of 72 hr, and a doubling time of approximately 4.5 hr. In FCS medium, titers increased 10(5)-fold over starting titers and reached a peak after 5 days of greater than 10(8) colony-forming-units (CFU)/ml. Optical density readings at 520 nm (OD(520)) served as useful estimates of the titers only during the last 30 hr of exponential growth. Before this time, titers were below 3 x 10(7) CFU/ml and could not be detected at OD(520). The growth-promoting activity of FCS appeared to be a normal serum component widely distributed among fetal calves. FCS from five commercial suppliers supported growth of R. quintana. The active factor(s) was: (i) non-dialyzable, (ii) resistant to heating at 56 C for 30 min, and (iii) partially inactivated at 100 C in 2 min and completely lost at 100 C in 10 min. The results emphasize the presence of erythrocyte and serum factors other than hemoglobin which stimulate the growth of R. quintana.     

Human lymphocyte cell cycle: Studies with the use of BrUdR

Summary The chronological distributions of human blood lymphocytes in first, second and third mitosis following PHA stimulation in vitro are presented. The first G₁ phase is shown to be of variable length resulting in some first mitoses appearing only about 150 h after stimulation. The serum or plasma used in the culture medium influences cell cycle time. A total cell cycle time of 10.6 h was estimated for cultures with autologous donor plasma and of 14.7 h for cultures with fetal calf serum. It was further calculated that following PHA stimulation 90% of the lymphocytes divide once, about 65% divide for a second and about 40% divide for a third time.     

Reaction of Chinese hamster cell culture to the cytotoxic action of imuran in the early and late stationary growth phases

It has been shown that when cells are incubated for a long time without medium change, imuran exerts a much greater effect on the viability of cells in the late stationary phase of growth than in the early stationary phase. When nutrient medium supplemented with 10% serum was changed for the medium with 0.5% serum, or when a partial change of medium (one third of the volume) was carried out daily from day 4, these differences were only slightly pronounced. Thus, the degree of cell response to imuran in the early and late stationary phases of growth depends upon the way of cell maintaining in the resting state.     

Changes in peroxidases in the suspension culture of Rubus fruticosus during growth

The growth parameters of a cell suspension culture of Rubus fruticosus L. were determined over a culture period including exponential growth, stationary phase and a glucose starvation period at the end of the normal culture cycle. Peroxidase activities were measured in the cytoplasm, in the cell wall, and in the culture medium by the guaiacol assay. There is a relationship between the activity found in the spent medium and the dry matter mass of the cells during the exponential growth. In the three compartments a bimodal repartition of peroxidase activities was observed, with the two peaks at day 4 and day 26, respectively. This suggests that the first peak corresponds to actively dividing cells whereas the second is associated with senescence, or stress due to starvation. Fractionation of the peroxidases from the culture mediuim revealed the presence of two sets of cationic isoenzymes, with minor amount of anionic peroxidases. Interestingly, the second peak of cationic enzymes which was of weak intensity at day 10 of the culture, becameprevalent at day 26. This indicates that not only the total amount of peroxidases varies as a function of culture time, but also that the nature of the peroxidases secreted into the medium changes during growth.Abbreviations DW dry weight - FW fresh weight - MV medium volume - SV suspension volume - BSA bovine serum albumin     

Bordetella pertussis agglutinogens in cultivation dynamics

Bordetella pertussis growth phases during homogenous batch dynamic cultivation in the liquid medium as well as during the static cultivation on the solid medium were established. The maximal activity of agglutination reaction with antisera to B. pertussis agglutinogens 1, 2, and 3 was detected in bacterial culture at the end of exponential phase of growth. The activity of agglutination reaction decreased when cultures in stationary and death phases were used. During transition from exponential to death phase level of antibodies to agglutinogen 2 decreased by4 - 32 times. 2 - 4-fold decrease of antibodies level was observed when antiserum to agglutinogen 3 was used. Activity of agglutination reaction with antiserum to agglutinogen 1 was high and did not depend from phase of growth. When polyvalent antiserum to B. pertussis was used 4-fold decrease of antibody titers was observed in parallel with change of growth phases. Sera from rabbits immunized with B. pertussis cultures from the middle of exponential growth phase, the end of this phase, and begin of the death phase had high (maximal) level of agglutinating antibodies (6400), which was detected on 101 day after immunization with the former culture and on 31 day after immunization with either of the two latter cultures. To the end of experiment (292 day) titers decreased to 800, 3200, and 1600 respectively. These findings confirm an advisability of use of exponential growth culture for immunization of rabbits in order to obtain highly active diagnostic antisera to B. pertussis.     

In vitro cultivation and behavior of aortic endothelium cells in a low serum culture medium

Endothelial cells isolated from calf aorta were used in the subculture No. 4 to 10 for experiments to establish standardized and well reproducible conditions of cultivation. The cells can be cultivated in the commercial medium Eagle-MEM with following supplements: 0.1 g L-glutamine, 5.0 g peptone, 0.5 g serum albumin, and 10 ml (= 1%) bovine serum per 1000 ml medium (MEMPAS). With the aid of immunofluorescence technique the cell type specific marker Factor VIII antigen was shown to be localized especially in the perinuclear region of the cells. The cells were characterized with regard to their growth behaviour. Both, the MEMPAS and the Eagle-MEM with 10 per cent serum increases the cell number in the first 4 days of the exponential growth to the same values. The use of MEMPAS in connection with a strict cultivation regime from the deep frozen cell suspension to culture in scintillation vials guaranteed well reproducible conditions of cultivation. In 14 non-selected experiments distributed over a longer period of time it was found that with regard to the values of the cell number on the respective day the cultures can be divided into two groups, which differ with statistical significance. In further experiments it was possible to confirm this result. Medium, conditioned by endothelial cells (K-MEMPAS) increases the cell number and the growth rate. From these results it was concluded that endothelial cells of vessels are able to produce growth factors with self-stimulating effects. At this time the endothelial cell line is stored in deep frozen state up to the 25th subculture. The endothelial cells cultivated in the described standardized conditions are useful for screening of cell type specific factors with angiogenic activity.     

Mitogenic activity of insulin in the culture of a trypanosome: duration and dose response

The effects of insulin on the in vitro growth characteristics of Trypanosoma granulosum were investigated. The insulin growth stimulus had a rapid onset with little or no lag phase but was relatively short lived, growth peaking typically on day 3 or 4 of culture. This contrasted with medium containing 10% foetal calf serum, normally used for in vitro culture of this trypanosome, which after a 3 day lag stimulated sustained log-phase growth. Insulin demonstrated a biphasic dose response with maximum growth typically occurring at 3-10 microg/ml and a much reduced effect at higher concentrations. These findings suggest a possible role for host insulin and/or trypanosomal insulin-like molecules in regulating parasite growth in vivo.     

Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperatures

AIMS: To quantify the influence of the growth phase, storage temperature and nutritional quality of the plate count medium on the apparent viability of Mannheimia haemolytica during storage at different temperatures. METHODS AND RESULTS: Mannheimia haemolytica was grown in shake flasks and in aerobic continuous culture to investigate factors affecting cell viability during storage, which was determined using plate counts on different media and epifluorescence microscopy. The high specific death rates of cells harvested after cessation of exponential growth and stored at 22, 4, -18 and -75 degrees C could be related to the rapid onset of exponential death in batch cultures. Yeast extract supplementation of the culture medium increased the viability of cells at most of the above-mentioned storage temperatures. Of the total cell count in continuous culture, only 48% could be recovered on brain-heart infusion agar, whereas supplementation of the agar medium with foetal calf serum increased the plate count to 71% of the total count. CONCLUSIONS: Mannheimia haemolytica cells harvested from the exponential growth phase had the highest survival rate during storage at low temperatures. Plate count values also depended on the nutritional quality of the agar medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Results presented here impact on the procedures for culture preservation and plate count enumeration of this fastidious animal pathogen.     

Arachidonic acid level in cellular lipids determines the amount of prostaglandins synthesized during cell growth in tissue culture

Methylcholanthrene transformed mouse fibroblast cells can be induced to synthesize prostaglandins by a short term incubation with various vasoactive agents including serum, bradykinin and thrombin or in response to mechanical detachment from the culture dish. The ability of the cells to synthesize prostaglandins upon stimulation changes during growth of the culture on the dish; the response is maximal on the first day after inoculation and decreased sharply thereafter. Feeding of the cells with fresh growth medium enhances prostaglandin production induced by all stimuli. The difference in the cell response during growth is probably not due to change of prostaglandin synthetase activity since the specific enzyme activities assayed with microsomal preparations of cells harvested from the first and third day culture are similar. However, analysis of the cellular content of arachidonic acid after saponification of the total lipid extract of cells harvested at different days of growth reveals that the level of arachidonic acid per cell during growth is parallel to the response to stimuli. It is maximal on the first day and decreases sharply on the second day and stays low on the third day. Our study suggests that the level of arachidonic acid in the cell governs the extent of prostaglandin synthesis upon stimulation.     

Arachidonic acid level in cellular lipids determines the amount of prostaglandins synthesized during cell growth in tissue culture.

Methylcholanthrene transformed mouse fibroblast cells can be induced to synthesize prostaglandins by a short term incubation with various vasoactive agents including serum, bradykinin and thrombin or in response to mechanical detachment from the culture dish. The ability of the cells to synthesize prostaglandins upon stimulation changes during growth of the culture on the dish; the response is maximal on the first day after inoculation and decreased sharply thereafter. Feeding of the cells with fresh growth medium enhances prostaglandin production induced by all stimuli. The difference in the cell response during growth is probably not due to change of prostaglandin synthetase activity since the specific enzyme activities assayed with microsomal preparations of cells harvested from the first and third day culture are similar. However, analysis of the cellular content of arachidonic acid after saponification of the total lipid extract of cells harvested at different days of growth reveals that the level of arachidonic acid per cell during growth is parallel to the response to stimuli. It is maximal on the first day and decreases sharply on the second day and stays low on the third day. Our study suggests that the level of arachidonic acid in the cell governs the extent of prostaglandin synthesis upon stimulation.     

5-Methylthioribose. Its effects and function in mammalian cells

The growth responses of 5-deoxy-5-methylthioribose on a 5'-deoxy-5'-methylthioadenosine phosphorylase containing cell line (BW5147) and the methylthioadenosine phosphorylase-deficient cell line (L1210D) were examined. Methylthioribose was shown to dramatically affect these cells, increasing their growth rate, saturation density, and viability. It was also found that methylthioribose could satisfy the methylthio dependence of the enzyme-deficient cell line, L1210D. A model is proposed to explain the selective growth of methylthioadenosine phosphorylase-deficient cells in medium lacking a methylthio donor but containing fetal calf serum. It is hypothesized that cellularly exported methylthioadenosine is degraded to methylthioribose in the presence of medium containing serum of high methylthioadenosine phosphorylase activity (i.e. fetal calf serum). The resultant methylthioribose can then be used to satisfy the methylthio requirement of these cells. To test this theory, various purified preparations of bovine liver methylthioadenosine phosphorylase were used to artificially increase the specific activity of methylthioadenosine phosphorylase in horse serum. In each case, it was demonstrated that only medium containing serum of enzyme activity nearly equal to that of the glutathione-stimulated fetal calf serum activity, supported the growth of methylthio-dependent cells in the absence of methylthio compounds. The data suggest that the degradation of methylthioadenosine and subsequent formation of methylthioribose represents an essential process in the growth of mammalian cells.     

Optimization of Conditions for Production of Channel Catfish Ovary Cells and Channel Catfish Virus DNA

Medium supplements were examined for their effect on the growth of channel catfish ovary cells. It was found that the usual serum supplement of 10% fetal calf serum could be successfully replaced with a combination of 5% fetal calf serum and a mixture of insulin, transferrin, and selenous acid. It was also found that these cells could be grown in a more efficient manner on microcarrier beads. This type of culture produced 14 times the number of cells per milliliter of total medium used compared with the usual tissue culture flasks used for cell growth. The microcarrier system also provided for greater production efficiency of DNA from channel catfish virus, a virus that infects this cell line.     

IgG production kinetics in serum free media

Summary A murine hybridoma (455) was cultured in four different serum free media formulations, and a newborn calf serum supplemented medium was used as a basis of comparison. The serum supplemented medium supports a higher cell growth rate and results in a higher IgG titer. However, the antibody secretion rate on a per cell basis is higher in the serum free media, indicating that serum could be inhibitory to antibody secretion. The results identify the possibility of a least eliminating serum during the monoclonal antibody production phase.     

Lambing rate using vitrified blastocysts is improved by culture with BSA and hyaluronan

Fatty acid-free bovine serum albumin (BSA(FAF)) can be added to supplement medium used in the culture of sheep embryos. BSA(FAF) was able to support blastocyst and subsequent embryo development at rates equivalent to that of fetal calf serum (FCS)-supplemented medium when fresh embryos were transferred. Furthermore, culture with BSA(FAF) significantly increased development of vitrified blastocysts transferred into synchronized sheep. The addition of the glycosaminoglycan, hyaluronan (HA) to the culture medium in the third and fifth day also increased cryo-tolerance of blastocysts and in turn lambing rate was improved.     

日本血吸虫尾蚴经人工方法转变的童虫体外培养的研究

本文介绍了日本血吸虫体外培养系统。建立了较适于童虫生长发育的B41培养基。尾蚴经人工方法转变的童虫在体外可发育至雌雄合抱,雌雄生殖器官形成。雌虫可达产卵阶段,但未具备正常的产卵机能。培养的血吸虫在体外至少可存活110天。     

Induction of hyaluronic acid synthetase activity in rat fibroblasts by medium change of confluent cultures

Hyaluronic acid synthesis in cultured cells usually occurs during the growth phase. The relation between hyaluronic acid synthetase activity and cell proliferation is studied. The synthetase activity in rat fibroblasts is high during the growth phase, but low in the stationary phase. When the old medium of stationary cultures is renewed with fresh medium containing 20% calf serum, DNA synthesis occurs synchronously between 12 and 20 hours, followed by cell division. Under these conditions, the hyaluronic acid synthetase activity is significantly induced within two hours, reaching a maximum level at 5–8 hours, and then decreases gradually. This induction of the synthetase, which shows a high turnover rate, requires continued synthesis of both RNA and protein. Furthermore, the induction of both DNA and hyaluronic acid synthesis is found to be caused by calf serum added in the medium. However, dialysis and ultrafiltration of the serum permit us to concentrate an active fraction with a high molecular weight, which induces the synthetase activity, but not DNA synthesis.     

Protein synthesis and secretion and RNA and DNA synthesis in human skin fibroblasts in a medium with a low serum content

Human skin fibroblasts, both postnatal and embryonic, were cultured in the stationary phase of growth for 6-10 days in the DMEM with bovine serum (BS), 0.1-0.5% fetal calf serum (FCS) or 1% human serum (HS). On the day 4 of culturing, a considerable increase was observed in the synthesis and secretion of protein by postnatal fibroblasts in the Eagle medium with 0.1-0.5% FCS, or with 0.5% BS, and in medium 199 with 0.1-0.5 BS, or with 0.1 FCS. Maximum synthesis and secretion of 14C-proline labeled protein was observed on day 2 of culturing of cells in the DMEM medium with 1% HS. In the DMEM medium with low serum content, protein synthesis being virtually unchanged, 75-80% of protein was secreted by cells into the culture medium with BS on days 2-4; in the medium with FCS such a high secretion of protein was observed only on day 4. High synthesis of protein by fetal fibroblasts in the DMEM medium with 0.1% BS and high protein secretion in all the media with 0.1% BS or 0.5% FCS were observed. The maximum level of secretion of protein by fibroblasts coincided with a considerable increase in both RNA and DNA syntheses. The data obtained suggest that cells in deep resting state actively react to the composition of the medium as well as to the quality and quantity of the serum. It may also be suggested that the mechanism of protein secretion has an important role in maintenance of the constant level of intracellular proteins in resting cells.     

Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content.

The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods. Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5%. A longer lag phase resulted when the lower serum content media were used. Cellular rates of glucose uptake showed a significant increase as serum levels were lowered. Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth. Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined. In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells. Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time. Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production. However, complete removal of serum from the medium resulted in a significant drop in antibody productivity. Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS. To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed. The growth rates were similar but there was a significant difference in IgG production rates. The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations. These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates. Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability.     

Amphibian cells in culture. I. Nutritional studies.

Nutritional requirements of amphibian cells in culture were studied for the purpose of modifying a minimal medium in which frog cells could proliferate and which could be used for obtaining drug-resistant and auxotrophic variants. The serum, purine, CO2, and amino acid requirements for ICR 2A (a Rana pipiens haploid cell strain) have been investigated employing two different media: L-15, a nonbicarbonate, amino acid-buffered medium and Eagle's MEM, a bicarbonate-buffered medium. In this paper we present evidence to support the following conclusions: (1) With L-15 as the base medium, 10% fetal calf serum (FCS) supports optimal cell growth during exponential phase. Calf serum, whole, dialyzed, or heat-inactivated, cannot substitute for FCS and, in fact, is inhibitory. (2) Purines are required by ICR 2A cells only if grown in a nonbicarbonate-buffered medium, since the cells under these conditions cannot produce enough endogenous CO2 to support de novo purine synthesis. (3) In addition to the amino acids considered essential for mammalian cells in culture, ICR 2A cells depend upon exogenous asparagine. Glutamine and/or aspartic acid cannot replace the asparagine requirement. However, ICR 2A cells do utilized exogenous glutamine as an oxidative substrate.     

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.