Studies on serum requirements for the cultivation of Plasmodium falciparum. 2. Medium enrichment

Previous experiments using RPMI 1640 medium have indicated that the dialysis of human serum removes components of low relative molecular mass (6000-8000 RMM) that are essential for continuous cultivation of Plasmodium falciparum. To determine which low-RMM components are important for parasite development, we compared growth in normal serum to that in dialysed serum using a number of other commercially available media, which we considered to be richer than RPMI 1640. Through these comparisons, we determined that hypoxanthine was the major dialysable nutrient required for parasite development. High quality bovine serum requires 3 - 12 × 10^-5 mol/litre of hypoxanthine as a supplement to support continuous cultures of P. falciparum. Thus far we have been unable to attain parasite growth in medium containing supplemented bovine serum that is as good as growth in medium containing human serum.     

Growth characteristics of canine pathogenic viruses in MDCK cells cultured in RPMI 1640 medium without animal protein

Madin Darby canine kidney (MDCK) cells were adapted to serum-free RPMI 1640 medium and used for cultivation of canine viruses. RPMI 1640 medium was supplemented with a soybean peptone, L-glutamine and antibiotics, so that the protein concentration was less than 5 microg/ml (RPMI/SP medium). The resulting adapted MDCK-SP cells showed steady growth after the twenty-eighth passage in RPMI/SP medium (MDCK-SP cell culture). Canine distemper virus, canine parvovirus, canine adenoviruses and canine parainfluenza virus, which are the principal components of canine combined virus vaccines, grew in the MDCK-SP cell culture as efficiently as the parental MDCK cells cultured in the conventional Eagle's MEM containing fetal bovine serum. Consequently, the use of MDCK-SP cell culture can make current canine vaccine products much safer, of higher quality and at lower cost.     

Stereomicroscopic and histological examination of bovine embryos following extended in vitro culture

Attempts to support survival of mammalian embryos after hatching have met with limited success, although some mouse studies have reported growth at the post-implantation stage. The aim of the present research was to establish and characterise an in vitro culture system that could support extended growth and differentiation of bovine embryos. Abattoir-derived oocytes were matured and fertilised in vitro. Presumptive zygotes were cultured in modified synthetic oviduct fluid (SOFaaci) medium supplemented with 5% cow serum (CS). On Day 9, single hatched blastocysts (n = 160) were randomly allocated to SOFaaci supplemented with either 5% bovine serum albumin, 5% CS, 5% fetal calf serum (FCS) or SOF only and cultured on a collagen gel substrate for up to 45 days. Embryos were evaluated at various time-points until complete disaggregation or the total disappearance of embryonic cells. Blastocyst viability post hatching was severely compromised in protein-free SOFaaci medium. Addition of FCS generated increased embryonic growth for the longest time period (Day 45) when compared to the other groups. Long-term survival of embryonic cells was observed stereomicroscopically by the proliferation and development of three-dimensional tubular structures to 85% confluence in culture. Haematoxylin and eosin staining of morphological structures obtained from all treatment groups revealed embryos displaying trophoblast, inner cell mass and hypoblast development to varying degrees. Regardless of treatment, extended in vitro culture did not result in development comparable with that described for in vivo embryos. In the present work, however, there was evidence of extended culture of bovine embryos beyond that achieved previously. However, further research is required to identify the exact requirements for extended in vitro culture for bovine embryos.     

Delayed culture of Leishmania in skin biopsies

Between January 1997 and October 1998, 16 skin biopsies collected from 13 patients with cutaneous leishmaniasis in French Guiana were inoculated in culture medium after travel for 3-17 days from the place of biopsy to the culture laboratory in France. Each biopsy fragment was introduced near the flame of a Bunsen burner into the transport medium (RPMI medium supplemented with 10% fetal calf serum) which was maintained at ambient temperature during postal delivery to France. In France the biopsies were ground in sterile saline before being inoculated into NNN culture tubes. The cultures were incubated at 25 degrees C and subcultured every week until the 5th week. The cultures were positive in 9 cases, remained negative in 4, and were contaminated in 3 cases. Positive results were obtained at all seasons and for 3 different Leishmania species. The study indicates that delayed culture can yield useful results from biopsies taken in field conditions.     

血清浓度对高原体外肝细胞培养的影响

目的了解在高原环境条件下血清对培养肝细胞生长的影响,初步确定进行体外肝细胞培养的适宜血清浓度。方法采用已适应本地环境的成年家兔肝细胞进行体外单层培养,在常规RPMI1640培养基的基础上添加不同浓度的新生牛血清(0%、2%、10%及20%),观察培养细胞形态、数量及培养上清中白蛋白和尿素水平变化。结果无血清组始终未见明显细胞贴壁及增殖,其他组细胞均于接种4 h开始贴壁,8 h开始增殖,10%血清组和20%血清组细胞均于6 d生长满瓶,并形成均匀的单层,而2%血清组细胞则于8 d生长满瓶;2%血清组的细胞数量峰值明显低于10%血清组和20%血清组;除无血清组外,培养上清中白蛋白和尿素水平均呈现显著升高,其中10%血清组和20%血清组的白蛋白及尿素峰值水平明显高于2%血清组。结论在高原环境条件下,体外培养肝细胞的生长、增殖及其生物学功能维持明显依赖于常规基础培养基中添加的血清浓度;对于普通肝细胞培养实验而言,10%血清浓度较为适宜。     

Hepatocytes as feeder-layers for in vitro cultivation of Plasmodium falciparum blood-stages

To improve the in vitro growth of Plasmodium falciparum we attempted to cultivate its erythrocytic stages on monolayers of functionally active hepatocytes. Hepatocytes from Swiss Albino mice were isolated by perfusing the liver with a collagenase solution and were co-cultured with a liver epithelial cell type in RPMI 1640 medium supplemented with 10% human umbilical cord serum. The results show that the presence of hepatocytes improves both the multiplication rates of three strains of P. falciparum already in cultivation and the proliferation of freshly isolated strains. Of nine primary isolates tested, only three could be adapted in the standard conditions, whereas all grew readily in the presence of hepatocytes. After two to three weeks of culture with feeder cells, all the strains could be maintained continuously in standard conditions. Similar results were obtained using hepatocytes from another rodent species. Growth was also improved using the supernatant from hepatocyte cultures. No improvement resulted from the use of two human hepatoma cell lines, one rat hepatoma, human embryonic lung fibroblasts, human liver fibroblasts and rat liver epithelial cells as feeder layers. From these results it appears that better culture media can be designed and that the effect of hepatocytes is probably related to the specific functions exhibited by these cells. Hepatocytes may act either by removing toxic substances, particularly lactic acid in the Krebs and Cori cycles, and/or supplying nutrients essential to the parasite.     

Maintenance of infectivity of Trypanosoma congolense in vitro with explants of infected skin at 37 degrees C

Glossina morsitans infected with two stocks of Trypanosoma congolense were fed on rabbits and calves to produce local skin reactions containing trypanosomes. Areas of infected skin were removed from the animals and used to prepare dermal explant cultures in Eagle's MEM and RPMI 1640 culture medium, supplemented with foetal bovine serum and containing penicillin and streptomycin. Cultures were incubated at 37 °C and media were changed at 24 to 48 hour intervals to maintain pH 7·0 to 7·2. There was evidence of trypanosome multiplication in explant cultures set up in both media; one trypanosome stock was maintained equally well in both Eagle's MEM and RPMI 1640, but the other stock survived better in Eagle's MEM. Explant cultures prepared from calf tissues generally yielded more trypanosomes at 24 hours than those prepared from rabbit tissues. The numbers of parasites present near the explants at 24 hours were maintained for up to 14 to 15 days before a decline in parasite concentration occurred. The organisms retained typical blood stream trypomastigote morphology and were infective for mice for periods up to 21 days. The trypanosomes growing in primary explant cultures could not be subpassaged in culture media alone or on to monolayers of fibroblast-like cells of bovine, murine or buffalo origin. Attempts to establish primary cultures by placing infected skin explants directly on to similar monolayers were also unsuccessful.     

A modification of Diamond's medium for the axenic culture of Entamoeba histolytica.

The use of casein hydrolysate in Diamond's axenic culture medium TPS-1 in replacement of trypticase allowed good growth of the trophozoites of Entamoeba histolytica. This modified medium also supported growth of trophozoites preserved for 16 months in liquid nitrogen. Considerable labour and cost of serum can be saved by using 5% instead of 10% bovine serum in combination with this modified medium.     

Plasmodium falciparum in vitro schizont maturation tests in Mozambique are not improved by removing immune parasite carriers' plasma

To study the effect of immune parasite carriers' plasma on Plasmodium falciparum schizont maturation, peripheral blood stages were incubated for 24-40 h in RPMI medium with either 5% carrier's plasma + 5% non-immune AB serum or 10% non-immune serum. The number of schizonts per 200 asexual P. falciparum was lower in non-immune serum than in the presence of carrier's plasma in 19 of 26 cases, due to increased frequency of schizont rupture when carrier's plasma was absent. It is concluded that, under these test conditions, the replacement of immune plasma by non-immune serum makes schizont maturation tests, which are based on the proportion of schizonts among asexual P. falciparum as a measure of growth, more difficult to interpret.     

Plasmodium falciparum in continuous culture: a new medium for the in vitro test for sulfadoxine sensitivity

The sulfadoxine sensitivity of two strains of Plasmodium falciparum from Thailand, FCM2 and FCM5, was assessed using two types of culture medium, Waymouth formula and RPMI 1640. Growth of the parasite was completely inhibited by 0.5 mmol of sulfadoxine per litre of Waymouth formula, whereas parasite growth in RPMI was not affected at this concentration. The apparent difference in drug sensitivity was shown to be caused by competition between 4-aminobenzoic acid and sulfadoxine. This hypothesis was further confirmed by the extent to which [¹⁴C]-sulfadoxine was incorporated into the infected erythrocytes.     

Role of testosterone,estradiol, and insulin in diet- and exercise-induced reductions in serum-stimulated prostate cancer cell growth in vitro

Prostate cancer risk is associated with a high-fat diet and a sedentary lifestyle. Placing men on a low-fat diet-and-exercise intervention reduces serum hormones, including estradiol, insulin, and free testosterone, that may play a role in prostate cancer growth. Eight men participated in a low-fat diet-and-exercise program for a mean of 14.2 yr, and LNCaP cell growth in culture was measured in medium supplemented with 10% of each subject's serum as well as with testosterone, estradiol, and insulin added singly or in combination. These results were compared in the fetal bovine serum (FBS)-stimulated growth and cell growth in serum obtained from a control group of 14 overweight men. In separate tissue culture experiments, LNCaP and PC-3 cell growth was also measured in response to the addition of testosterone, estradiol, or insulin to steroid-stripped FBS. LNCaP cell growth in medium with subject serum was 40% less than in FBS-stimulated medium and 49% less than in medium with serum from control, overweight men. Addition of testosterone, estradiol, and insulin to serum from diet-and-exercise subjects significantly stimulated LNCaP cell growth in vitro but accounted for only about half of the difference between the control and diet-and-exercise subjects. Thus other serum changes must also account for the significant reduction in LNCaP cell growth observed using medium with serum from the diet-and-exercise subjects in the cell culture assay.     

Observations on adult Onchocerca volvulus maintained in vitro

Adult Onchocerca volvulus were enzymatically isolated with collagenase from excised nodules and kept in TC medium 199 with Hank's salts supplemented with various sera. Male and female worms survived in the culture medium and 10% human serum on average for about 11 days (maximum 28 days) and 14.5 days (maximum 42 days). Up to 4,000 microfilariae were expelled by each female per day, but the production of new oocytes or development of embryos could not be observed in vitro.     

Lack of an association between cellular phospholipid triene: tetraene ratio and proliferation of human skin fibroblasts in culture

Two experiments were performed in an attempt to establish an association between cellular phospholipid triene:tetraene ratio and proliferation of human neonatal skin fibroblasts in culture. In Experiment 1, a low lipid culture medium was developed that caused an accumulation of (n-9) eicosatrienoic acid in the phospholipids of human fibroblasts. This culture medium, when supplemented with a mixture of mitogens, supported growth of human fibroblasts at a level equivalent to that found under conditions of maximal growth using serum supplementation (8% fetal bovine serum). The triene:tetraene ratio of fibroblast phospholipids under the two conditions was 1.88 vs. 0.03, suggesting that the growth of these cells was not adversely affected by a high (greater than 0.4) triene: tetraene ratio. In Experiment 2, cells were cultured in a low lipid, mitogen-supplemented medium with 16:1(n-7), 18:1(n-9), 18:2(n-6) or 20:4(n-6) added as the albumin complex. All the fatty acids permitted an equivalent maximal growth stimulation in the assay system, although having different effects on the phospholipid triene:tetraene ratio. The results suggest that there is a lack of an association between cellular phospholipid triene:tetraene ratio (range, 0.03 to 3.4) and proliferation of human fibroblasts in this culture system.     

Uptake of exogenous and endogenous eicosapentaenoic acid by cultured human mononuclear cells

1. Peripheral blood mononuclear cells from eight healthy volunteers were cultured, with or without concanavalin A (Con A), in a medium containing (ml/l) 100 normal autologous serum, 100 experimental autologous serum or 100 heterologous (fetal calf) serum. 2. The control and experimental autologous sera were obtained from the volunteers, before and after 15 d supplementation with 15 g fish oil (MaxEPA)/d to provide 1.5 g eicosapentaenoic acid (EPA; 20:5n-3)/d. The sera were frozen at -20 degrees. The level of EPA increased from trace quantities in the control autologous serum to 14.3% w/w free fatty acids and between 6.9 and 8.1% w/w lipoprotein phospholipids in the experimental autologous serum. The heterologous fetal calf serum was enriched with EPA, complexed with bovine serum albumin, to provide a final concentration of 15 micrograms/ml. All culture medium contained 10 ml fresh autologous serum/l and cells were obtained from the volunteers for the culture studies about 60 d after the end of EPA supplementation. 3. Portions of cells were removed from culture at 36, 48 and 72 h for phospholipid fatty acid analysis. 4. The level of EPA in phospholipids of cells cultured with exogenous EPA in fetal calf serum was increased significantly (P less than 0.05) at all sampling times, both with and without Con A. By 48 h the levels had peaked at 15.8 (SE 2.7) and 18.4 (SE 4.5)% w/w respectively. 5. Resting cells, i.e. with no Con A present, failed to accumulate EPA when cultured in the experimental autologous serum containing 8.6% w/w total lipids as endogenous EPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lycopene on prostate LNCaP cancer cells in culture

Epidemiological studies have shown an inverse relationship between serum lycopene levels and the risk of prostate cancer. The objective of this study was to measure the effect of lycopene on the proliferation of LNCaP human prostate cancer cells in culture. A new, water-dispersible lycopene in an appropriate vehicle was used. The stock solution was diluted in the medium to obtain lycopene concentrations of 10(-6), 10(-5), and 10(-4) M; their corresponding vehicles were similarly diluted to be used as controls. Cells were grown for 48 hours in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics. Lycopene was then added at different concentrations, and the cells were allowed to grow for 24, 48, 72, and 96 hours. Lycopene at concentrations of 10(-6) and 10(-5) M significantly reduced the growth of LNCaP cells after 48, 72, and 96 hours of incubation, by 24.4% to 42.8% (P <.05). The inhibitory effect of lycopene was significantly higher than that of the corresponding vehicle controls. In a follow-up experiment, a lower range of lycopene concentrations (10(-9) to 10(-7) M) was used to determine whether there was a dose-response effect. Lycopene significantly decreased the growth of cells in a dose-dependent manner when cells were incubated for 24, 48, 72, or 96 hours (F = 3.150, 11.27, 54.51, and 297.5, respectively; P <.05). The growth inhibitory effect of lycopene on human prostate cancer cells observed in this study suggests a possibly important role for lycopene as an antioxidant in human prostate cancer; however, investigations of other mechanisms are warranted.     

混合血清法筛检丙型肝炎抗体的前瞻性研究

为了解在实际检测条件下，混合血清法筛检丙型肝炎抗体的效果，对１８７５例献血员进行了前瞻性研究。在单个血清检测结果未知的条件下，将每５个血清混合，使用酶免疫法检测丙型肝炎抗体。在检测中保持混合血清中每个标本的稀释度与单个血清检验法相同。结果显示：本次抗－ＨＣＶ筛检的血清阳性率为２．２４％，以单个血清检验为对照，混合血清法的灵敏度为１００％，特异度为９９．２％；阴性混合标本的ＯＤ／ＣＯ值呈正偏态分布，当混合标本中含有高ＯＤ值阴性标本时，可产生假阳性结果。收益分析发现：应用混合血清法筛检抗－ＨＣＶ可节省６９．３３％的检测费用。     

A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions

Rabies virus strain production in Vero cells grown on Cytodex 1 in a 2 L stirred tank bioreactor and in a medium free of components of human or animal origin (VP-SFM) is described. Cell banking procedure in VP-SFM supplemented with an animal components free mixture (10%DMSO+0.1%methylcellulose) was reported and cell growth after revitalization was assessed. Vero cells exhibited growth performances (specific growth rate and cell division number) similar to that obtained in serum containing medium. To design a scalable process that is totally free of animal-derived substances, two proteases: TrypLE Select and Accutase, were assessed as an alternative to trypsin which is routinely used for cell passage. Growth performance of Vero cells grown in VP-SFM and MEM+10% fetal calf serum (FCS) over four passages and subcultivated with either TrypLE Select or Accutase was evaluated. TrypLE Select showed the best performance in terms of specific growth rate and cell division number. Kinetics of cell growth and rabies virus production (LP2061/Vero strain) were investigated in spinner flask and in a 2 L bioreactor. In spinner flask, a maximal cell density level of 1.85x10(6) cells/mL was achieved when the cells were grown in VP-SFM on 2 g/L Cytodex 1. Cell infection experiments conducted at an MOI of 0.3 and without the medium exchange step, typically needed for serum containing rabies virus production, resulted in a maximal virus titer equal to 2x10(7) (Fluorescent Focus Unit) FFU/mL. In stirred tank bioreactor, Vero cell growth in VP-SFM on 3 g/L Cytodex 1 was shown to be sensitive to the aeration mode. Sparging the culture was detrimental for cell growth, whereas cell density level was greatly enhanced when only headspace aeration was used. A cell density level of 2.6x10(6) cells/mL was obtained when the cells were grown on 3g/L Cytodex 1 and in batch culture mode. Cell infection at an MOI of 0.1 without any medium exchange, yielded a maximal rabies virus titer of 2.4x10(7) FFU/mL. Furthermore, Vero cell growth in a 2 L bioreactor using recirculation culture mode during cell proliferation step and perfusion for virus multiplication phase was investigated. In comparison to batch culture, a higher cell density level that was equal to 5x10(6) cells/mL was reached. Cell infection under conditions similar to batch culture, resulted in a maximal virus titer equal to 1.38x10(8) FFU/mL. The potency of the pooled inactivated virus harvests showed an activity of 2.58 IU/mL which was comparable to that obtained in serum supplemented medium.     

盐亭食管癌高发区饮食对人食管癌细胞增殖的影响

目的采用血清生理学方法,观察盐亭食管癌高发区饮食喂饲大鼠血清对人食管癌细胞Eca-109生长增殖的影响。方法将24只雄性SD大鼠分为3组,分别喂饲大鼠常规饲料、健康成人饮食及盐亭饮食7天,每天定时记录采食量及体重。用MTT法探讨用大鼠血清培养人食管癌细胞的适宜条件;分别以人正常肝上皮细胞HL7702及10%灭活小牛血清作为对照,采用细胞生长曲线、细胞群体倍增时间、3H-TDR掺入实验研究盐亭饮食喂饲大鼠血清对人食管癌细胞生长增殖的影响。结果喂饲健康成人饮食与常规饲料的大鼠其采食量及体重无差异;用5%未灭活大鼠血清取代10%灭活小牛血清适合人食管癌细胞培养;盐亭食管癌高发区饮食喂饲大鼠血清可明显促进人食管癌细胞生长,却不利于人正常肝上皮细胞生长,且与其他3个组均有统计学差异(P<0.05)。结论盐亭食管癌高发区饮食具有促进人食管癌细胞Eca-109生长增殖的作用。     

A rapid in vitro assay system using anti-bromodeoxyuridine for drug susceptibility of Plasmodium falciparum

Drug effects were monitored by the measurement of bromodeoxyuridine (BrdU) incorporation into parasite deoxyribonucleic acid (DNA). Cells are pulse-labelled with BrdU and those which are synthesizing DNA incorporate BrdU into their DNA. Anti-BrdU is used to identify cells undergoing DNA synthesis at the time of the pulse. Concentration-effect curves were determined for chloroquine, pyrimethamine and quinine in culture for only 3 h in RPMI 1640 medium supplemented with BrdU. Different strains of Plasmodium falciparum could be distinguished when BrdU uptake was monitored by the ELISA. This assay offers a fast and accurate method of monitoring the effects of a variety of antimalarial drugs.     

Porcine reproductive and respiratory syndrome antibody detection on filter discs

Two enzyme-linked immunosorbent assay (ELISA) kits were evaluated for detection of porcine reproductive and respiratory syndrome (PRRS) antibodies in pooled sera and filter discs (FDs). Elution and incubation procedures and positive thresholds for both ELISAs were determined using FDs collected from sixty non-infected pigs and five pigs with low PRRS-antibody titres. Eighty paired samples (serum/FD) from infected pigs were titrated using both ELISAs. The authors thus showed that five sera or five FDs could be pooled in one test without significant loss of sensitivity. Compared to individual sera, method sensitivity was found to be 79% and specificity 97.5%, based on data from 200 pools of FDs collected on 15 PRRS-infected farms and 120 pools collected on 71 non-infected farms. To balance loss of sensitivity, two pools of five samples from sows and one pool of five samples from finishing pigs can be tested as an alternative to seven and five single sera, respectively.