Leishmania (Viannia) braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania) amazonensis

We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania) amazonensis replication rates were similar in both tissue culture media and reached maximum rates by 48 h. In contrast Leishmania (Viannia) braziliensis growth was significantly greater in TC-100 but maximum rates were achieved by 96 h. Folic acid appears to be the limiting factor and supplementation of Schneider media with this nutrient improved L. (V.) braziliensis replication rates and decreased the time of maximum replication to 48 h.

More productive in vitro culture of Cryptosporidium parvum for better study of the intra- and extracellular phases

The great difficulties in treating people and animals suffering from cryptosporidiosis have prompted the development of in vitro experimental models. Due to the models of in vitro culture, new extracellular stages of Cryptosporidium have been demonstrated. The development of these extracellular phases depends on the technique of in vitro culture and on the species and genotype of Cryptosporidium used. Here, we undertake the molecular characterization by polymerase chain reaction-restriction fragment lenght polymorphism of different Cryptosporidium isolates from calves, concluding that all are C. parvum of cattle genotype, although differing in the nucleotide at positions 472 and 498. Using these parasites, modified the in vitro culture technique for HCT-8 cells achieving greater multiplication of parasites. The HCT-8 cell cultures, for which the culture had not been renewed in seven days, were infected with C. parvum sporozoites in RPMI-1640 medium with 10% IFBS, CaCl2 and MgCl2 1 mM at pH 7.2. Percentages of cell parasitism were increased with respect to control cultures (71% at 48 h vs 14.5%), even after two weeks (47% vs 1.9%). Also, the percentage of extracellular stages augmented (25.3% vs 1.1% at 96 h). This new model of in vitro culture of C. parvum will enable easier study of the developmental phases of C. parvum in performing new chemotherapeutic assays.

In vitro culture of Spondias mombin L. nodal segments

Spondias mombin L. shoot cultures were initiated from nodal explants taken from plants propagated by seeds. Explants coming from 4-6 months old plants, previously disinfected, were cultivated on WPM medium supplemented with a wide range of concentrations of BAP (0.0, 0.22, 0.44, 2.22 and 4.44 muM) and NAA (0.0, 0.27 and 2.70 muM). After four weeks, the responses obtained were axillary shoot and root formation. The first response were preferentially induced with the medium containing only BAP, regardless of the BAP concentration. The addition of NAA on medium reduced significantly axillary shoot formation and induced rhizogenesis. Roots were formed on nodal explant basis, preferentially on medium supplemented with 4.44 muM NAA. The medium supplemented with BAP reduced significantly root formation.

In vitro culture of Phyllanthus stipulatus (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus stipulatus (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication rates (8-9 shoots per explant) was achieved on MS media supplemented with either 2.5-5.0 muM IBA. The best basal media for axillary shoot proliferation when 0.62 muM BA was supplemented were MS, MS/2 and AR (4-5 shoots per explant). Rooting was achieved with 100% of the microshoots on MS medium without growth regulators. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed in 81% of the ex vitro grown plantlets after 12 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated either in the vertical position, in the light on MS medium supplemented with 5.0 muM NAA or horizontally oriented, in the dark on MS supplemented with 5.0 muM NAA or 1.25-5.0 muM BA or 2iP. Root cultures were successfully established on MS medium containing 1.1 muM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/organ culture techniques for vegetative propagation and secondary metabolism studies.

In vitro culture at low temperature and ex vitro acclimatization of Vriesea inflata an ornamental bromeliad

(In vitro culture at low temperature and ex vitro acclimatization of Vriesea inflata an ornamental bromeliad). In vitro culture by seeds is a technique for preservation of threatened species because it may provide a large number of plants with genetic diversity. The bromeliad Vriesea inflata (Wawra) Wawra, an ornamental bromeliad, is extensively and illegally collected from the nature and must be preserved. It is possible to form plant threatened collections in vitro by reducing the temperature of culture, while occupying little space, with the consequent reduction of maintenance costs. This work evaluated the influence of temperature on in vitro growth and morphology of plants of V. inflata, with the aim of establishing a slow growth-rate and analyzing the ex vitro acclimatization. Seeds were germinated in vitro in Murashige and Skoog (MS) medium, with macronutrients reduced to 50% (MS/2). After three months the plants were transferred to flasks of new same medium and kept in two germination chambers with the temperature adjusted to 15 °C and to 28 °C. After 24 months the plants were subject to biometric, photosynthetic pigments content and anatomical analyses. Results showed that plants maintained at 15 °C were smaller than those at 28 °C. Nevertheless, there were no alterations in pigments content, anatomy. In both treatments there was a survival rate of 100%. This work showed that plants of this species can be kept in vitro at 15 °C with the aim of forming a slow-growth collection, thereby seeking its preservation, and can be transferred to growth at ex vitro condition to achieved 100% survival rate.

Intra and interspecific variability of in vitro culture response in Lycopersicon (tomatoes)

Intra and interspecific variability was measured in the genus Lycopersicon for the traits: productivity rate (PR, total number of regenerated shoots/total number of cultures), regeneration percentage (%R, number of cultures regenerating shoots or primordia/total number of cultures) and callus percentage (%C, number of cultures only producing callus/total number of cultures). Leaf explants from various genotypes of L. esculentum, L. esculentum var. cerasiforme, L. pimpinellifolium and L. peruvianum were placed on Murashige and Skoog (Physiol. Plant. 15: 473-493, 1962) medium + 0.175 mg/l IAA + 2.25 mg/l BA. Significant differences among species and among genotypes within the same species were found, while genotypes from different species showed similar responses.

Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

Oviposition by Schistosoma mansoni during in vitro cultivation

Observation of Schistosoma mansoni oviposition during in vitro culture of adult worms for a maximum period of 10 days showed three well distinct phases in the kinetics of oviposition: an initial phase with low egg production, a period of maximum oviposition and finally a progressive reduction in the number of eggs during the late phases of culture. The kinetics of oviposition and the number of eggs laid by the parasites are influenced by the number of worm pairs per amount of RPMI 1640 medium, time of parasite development in the vertebrate host and type of serum utilized in the culture medium.

Study of some parameters affecting the in vitro cultivation of Plasmodium falciparum within saimiri sciureus red blood cells

The in vitro growth and multiplication of the erythrocytic stages of Plasmodium falciparum within Saimiri sciureus (squirrel monkey) red blood cells have been studied. Various parameters, such as the origin of the red blood cells and serum supplement, nature of the buffer, influence of the final pH of the medium, role of proteose peptone and glucose addition, were investigated. The selection of the best culture conditions led to the obtention of a reproducible in vitro growth of two parasite cycles in Saimiri erythrocytes, which is an useful achievement for in vitro studies. Our failure to establish a continuous culture line for longer than 19 days, could be explained by a dramatic increasing of osmotic fragility of the Saimiri red blood cells related to their small size.

Ultrastructural study of the TG180 murine sarcoma cell invasion by Toxoplasma gondii: comparison between in vivo and in vitro cell cultures

Infection of non-adherent TG180 murine sarcoma cells with Toxoplasma gondii was compared, at the ultrastructural level, in both in vivo and in vitro conditions. Suspensions of 3.0 x 10(6) TG180 cells infected in vitro with 1.0 x 10(6) parasites of the RH strain were harvested between the first and 6th day post-infection and processed for transmission electron microscopy. In vivo infection was made by intraperitoneal inoculation in mice of 1.0 x 10(6) TG180 cells, that were co-inoculated with a parasite suspension at the same cell concentration. Cells were harvested 10, 20, 30 min and 24, 48 h post-inoculation and processed for transmission electron microscopy at the same conditions of the in vitro culture. It was observed TG180 murine sarcoma cells with intense and equivalent intracellular parasitism in both conditions. Host cells with parasitophorous vacuoles containing up to 16 parasites, as well as parasites undergoing mitoses or presenting a bradyzoite-like morphology, were frequently seen in both culture methods.

In vitro effects of amodiaquine on paired Schistosoma mansoni adult worms at concentrations of less than 5 µg/mL

In this study, the in vitro effects of amodiaquine (AQ) monotherapy on the egg output of paired adult Schistosoma mansoni worms and their survival during in vitro culture were assessed. In addition, the gross morphological alterations of male and female worms caused by AQ were visually observed under a dissecting microscope. AQ significantly reduced the daily egg output of paired adult S. mansoni worms following incubation for 14 days at 1-5 µg/mL, but not at 0.5 µg/mL, compared with the control group. AQ also reduced the survival of male and female worms at concentrations of 2 and 5 µg/mL, respectively. Moreover, exposure to 5 µg/mL AQ caused severe swelling and/or localisation of black content in the body of all male and female worms within one or two days of incubation; subsequently, shrinkage in the male worms and elongation in the female worms were observed. The initial morphological alterations caused by AQ occurred along the intestinal tract of the male and female worms. To our knowledge, this is the first study to report not only the efficacy of AQ at concentrations lower than 5 µg/mL on paired adult S. mansoni worms, but also the effects of AQ on the intestinal tracts of worms in in vitro culture.

In vitro organogenesis from internodal segments of adult sweet orange plants

The objective of this work was to optimize in vitro plant regeneration via organogenesis from tissues of adult 'Hamlin', 'Pêra', and 'Valência' sweet orange plants. Explants were grown in EME culture medium with different concentrations of 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA), at 27ºC in the absence of light for 50 days, followed by a 16-hour photoperiod for 20 days. Regeneration was assessed 50 and 70 days after in vitro culture. Organogenesis in cultivars Hamlin and Valência was promoted by EME supplemented with BAP, while NAA showed no apparent effect.

Residual effect of growth regulators in etiolation and regeneration of in vitro pineapple plants

This work aimed to evaluate the influence of naphthaleneacetic acid (NAA) and gibberellic acid (GA3) plant regulators in in vitro etiolation and subsequent regeneration of the PE x SC-60 pineapple hybrid. Nodal segments of in vitro plants with approximately 5-7 cm height were incubated in basic MS culture medium supplemented with 0.0; 0.5 and 1.0 mg L-1 of naphthaleneacetic acid (NAA) in combination with gibberellic acid (GA3) in concentrations of 0.0; 0.5 and 1.0 mg L-1, and maintained at 27 ºC under dark condition. Evaluations were carried out at 90 and 180 days after incubation period. The best results for length of etiolated stems were obtained with 1.0 mg L-1 of NAA. In the experiment followed by the regeneration, stems with 3 cm from the etiolation treatment, were cultivated in proliferation medium and the number of regenerated plants per treatment was evaluated at 60 days of cultivation. The treatment that promoted the best etiolation of plants also promoted the worst regeneration rates, demonstrating the residual effect of the auxin used in the previous step in the regeneration of plants of the pineapple hybrid evaluated.

Metabolic response induced by endophytic fungi and bacteria in H. marrubioides Epling in vitro microplants

Hyptis marrubioides Epling is a native plant from Brazilian Cerrado. In this paper, the response of in vitro microplants of this species to inoculation with bacterial and fungal endophytic isolates is evaluated. HPLC-DAD analysis showed the presence of 3,4-O-(Z)-dicaffeoylquinic acid and quercetin-7-O-glucoside as the main components. GC/MS analysis demonstrated that the sesquiterpenes τ-cadinol and caryophyllene oxide were only produced in microplants inoculated with endophytic bacteria, while methyl hexadecanoate, methyl heptadecanoate and methyl (Z,Z,Z) 9,12,15-octadecatrienoate and the triterpene methyl 3β-hydroxy-urs-12-en-28-oate were overexpressed only when the microplant was treated with endophytic fungi.