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.

Genetics of resistance to the fungus Helminthosporium sativum in wheat: use of culture filtrates in tissue culture

Six wheat genotypes and their F1 and F2 generations were exposed to the action of Helminthosporium sativum culture filtrates to examine the genetics of hexaploid wheat resistance. The objective was to improve the efficiency of breeding programs by identifying the action and number of genes involved in the resistance. The varied response of the tested genotypes to the culture filtrates allowed division of the genotypes into four groups: resistant, moderately resistant, moderately susceptible and susceptible. This variability was detected in the progeny, suggesting that the parents have distinct genetic constitutions. Additive gene action predominated and genetic gain was shown to be possible through selection. The genetic control of the resistance trait seems to be complex because of the presence of gene interaction and the difficulty of eliminating the environmental effects. The inheritance seems to be oligogenic

Changes in N2 fixation in Stylosanthes scabra derived from tissue culture

Plants were regenerated from leaf-derived callus culture of Stylosanthes scabra, a polyploid legume tolerant to drought and adapted to acid soils. A total of 168 regenerants were planted out in Leonard jars in a complete randomized design. Nitrogen fixation and vegetative growth were indirectly evaluated by shoot dry weight, root dry weight, shoot N content and acetylene reduction activity. The results showed higher variation in the regenerants than in controls not submitted to tissue culture. Significant differences were found for all nitrogen fixation related-traits

Effect of actinomycin D on simian rotavirus (SA11) replication in cell culture

Rotaviruses are the major cause of viral diarrhea in humans and animals. Actinomycin D (Act D) is an antibiotic that intercalates DNA and therefore inhibits DNA-dependent transcription. The current study was carried out to assess the influence of Act D on the replication of simian rotavirus (SA11) in cell culture. Virus-infected MA-104 cell cultures were studied in the presence of Act D at concentrations of 1.25 and 2.5 µg/ml. Treatment of rotavirus-infected cells with 2.5 µg/ml Act D 48 h post-infection reduced the cytoplasmic metachromasia after staining with acridine orange by 25%. Viral RNA labeled with ³H-uridine in the presence of the drug was separated by polyacrylamide gel electrophoresis. Viral RNA replication was not affected by Act D, but increased ³H-uridine uptake was demonstrable by infected cells in the presence of the drug. This possibly was due to the inhibition of cellular RNA synthesis by Act D, which thus enhances incorporation of the radionuclide into the viral RNA. Act D reduced the number of infected cells presenting virus-specific fluorescence 48 h post-infection by more than 50%. These data suggest that Act D may have complexed with viral RNA and prevented newly synthesized mRNA from being translated, but may not have prevented early replication.

Correlation of mixed lymphocyte culture with chronic graft-versus-host disease following allogeneic stem cell transplantation

The purpose of the present study was to evaluate the mixed lymphocyte culture as a predictive assay of acute and chronic graft-versus-host disease (GVHD). We studied 153 patients who received a first bone marrow transplantation from human leukocyte antigen-identical siblings. Acute GVHD was observed in 26 of 128 (20.3%) patients evaluated and chronic GVHD occurred in 60 of 114 (52.6%). One-way mixed lymphocyte culture (MLC) assays were performed by the standard method. MLC results are reported as the relative response (RR) from donor against patient cells. The responses ranged from -47.0 to 40.7%, with a median of 0.5%. The Kaplan-Meier probability of developing GVHD was determined for patients with positive and negative MLC. There was no significant difference in incidence of acute GVHD between the groups studied. However, the incidence of chronic GVHD was higher in recipients with RR >4.5% than in those with RR <=4.5%. The Cox Proportional Hazards model was used to examine the effect of MLC levels on incidence of chronic GVHD, while adjusting for the potential confounding effect of others suspected or observed risk factors. The relative risk of chronic GVHD was 2.5 for patients with positive MLC (RR >4.5%), 2.9 for those who received peripheral blood progenitor cells as a graft, and 2.2 for patients who developed previous acute GVHD. MLC was not useful for predicting acute GVHD, but MLC with RR >4.5% associated with other risk factors could predict the development of chronic GVHD, being of help for the prevention and/or treatment of this late complication.

Isolation and culture of umbilical vein mesenchymal stem cells

Bone marrow contains a population of stem cells that can support hematopoiesis and can differentiate into different cell lines including adipocytes, osteocytes, chondrocytes, myocytes, astrocytes, and tenocytes. These cells have been denoted mesenchymal stem cells. In the present study we isolated a cell population derived from the endothelium and subendothelium of the umbilical cord vein which possesses morphological, immunophenotypical and cell differentiation characteristics similar to those of mesenchymal stem cells isolated from bone marrow. The cells were isolated from three umbilical cords after treatment of the umbilical vein lumen with collagenase. The cell population isolated consisted of adherent cells with fibroblastoid morphology which, when properly stimulated, gave origin to adipocytes and osteocytes in culture. Immunophenotypically, this cell population was found to be positive for the CD29, CD13, CD44, CD49e, CD54, CD90 and HLA-class 1 markers and negative for CD45, CD14, glycophorin A, HLA-DR, CD51/61, CD106, and CD49d. The characteristics described are the same as those presented by bone marrow mesenchymal stem cells. Taken together, these findings indicate that the umbilical cord obtained from term deliveries is an important source of mesenchymal stem cells that could be used in cell therapy protocols.

Cholesterol induces fetal rat enterocyte death in culture

The effect of cholesterol on fetal rat enterocytes and IEC-6 cells (line originated from normal rat small intestine) was examined. Both cells were cultured in the presence of 20 to 80 µM cholesterol for up to 72 h. Apoptosis was determined by flow cytometric analysis and fluorescence microscopy. The expression of HMG-CoA reductase and peroxisome proliferator-activated receptor gamma (PPARgamma) was measured by RT-PCR. The addition of 20 µM cholesterol reduced enterocyte proliferation as early as 6 h of culture. Reduction of enterocyte proliferation by 28 and 41% was observed after 24 h of culture in the presence and absence of 10% fetal calf serum, respectively, with the effect lasting up to 72 h. Treatment of IEC-6 cells with cholesterol for 24 h raised the proportion of cells with fragmented DNA by 9.7% at 40 µM and by 20.8% at 80 µM. When the culture period was extended to 48 h, the effect of cholesterol was still more pronounced, with the percent of cells with fragmented DNA reaching 53.5% for 40 µM and 84.3% for 80 µM. Chromatin condensation of IEC-6 cells was observed after treatment with cholesterol even at 20 µM. Cholesterol did not affect HMG-CoA reductase expression. A dose-dependent increase in PPARgamma expression in fetal rat enterocytes was observed. The expression of PPAR-gamma was raised by 7- and 40-fold, in the presence and absence of fetal calf serum, respectively, with cholesterol at 80 mM. The apoptotic effect of cholesterol on enterocytes was possibly due to an increase in PPARgamma expression.

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.

Use of blends of bioabsorbable poly(L-lactic acid)/poly(hydroxybutyrate- co-hydroxyvalerate) as surfaces for Vero cell culture

Vero cells, a cell line established from the kidney of the African green monkey (Cercopithecus aethiops), were cultured in F-10 Ham medium supplemented with 10% fetal calf serum at 37°C on membranes of poly(L-lactic acid) (PLLA), poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and their blends in different proportions (100/0, 60/40, 50/50, 40/60, and 0/100). The present study evaluated morphology of cells grown on different polymeric substrates after 24 h of culture by scanning electron microscopy. Cell adhesion was also analyzed after 2 h of inoculation. For cell growth evaluation, the cells were maintained in culture for 48, 120, 240, and 360 h. For cytochemical study, the cells were cultured for 120 or 240 h, fixed, processed for histological analysis, and stained with Toluidine blue, pH 4.0, and Xylidine ponceau, pH 2.5. Our results showed that cell adhesion was better when 60/40 and 50/50 blends were used although cells were able to grow and proliferate on all blends tested. When using PLLA/PHBV (50/50) slightly flattened cells were observed on porous and smooth areas. PLLA/PHBV (40/60) blends presented flattened cells on smooth areas. PLLA/PHBV (0/100), which presented no pores, also supported spreading cells interconnected by thin filaments. Histological sections showed that cells grew as a confluent monolayer on different substrates. Cytochemical analysis showed basophilic cells, indicating a large amount of RNA and proteins. Hence, we detected changes in cell morphology induced by alterations in blend proportions. This suggests that the cells changed their differentiation pattern when on various PLLA/PHBV blend surfaces.

Immunophenotype of hematopoietic stem cells from placental/umbilical cord blood after culture

Identification and enumeration of human hematopoietic stem cells remain problematic, since in vitro and in vivo stem cell assays have different outcomes. We determined if the altered expression of adhesion molecules during stem cell expansion could be a reason for the discrepancy. CD34+CD38- and CD34+CD38+ cells from umbilical cord blood were analyzed before and after culture with thrombopoietin (TPO), FLT-3 ligand (FL) and kit ligand (KL; or stem cell factor) in different combinations: TPO + FL + KL, TPO + FL and TPO, at concentrations of 50 ng/mL each. Cells were immunophenotyped by four-color fluorescence using antibodies against CD11c, CD31, CD49e, CD61, CD62L, CD117, and HLA-DR. Low-density cord blood contained 1.4 ± 0.9% CD34+ cells, 2.6 ± 2.1% of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beads and cultured for up to 7 days. The TPO + FL + KL combination presented the best condition for maintenance of stem cells. The total cell number increased 4.3 ± 1.8-fold, but the number of viable CD34+ cells decreased by 46 ± 25%. On the other hand, the fraction of CD34+CD38- cells became 52.0 ± 29% of all CD34+ cells. The absolute number of CD34+CD38- cells was expanded on average 15 ± 12-fold when CD34+ cells were cultured with TPO + FL + KL for 7 days. The expression of CD62L, HLA-DR and CD117 was modulated after culture, particularly with TPO + FL + KL, explaining differences between the adhesion and engraftment of primary and cultured candidate stem cells. We conclude that culture of CD34+ cells with TPO + FL + KL results in a significant increase in the number of candidate stem cells with the CD34+CD38- phenotype.

Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture

The visual system is a potential target for methylmercury (MeHg) intoxication. Nevertheless, there are few studies about the cellular mechanisms of toxicity induced by MeHg in retinal cells. Various reports have indicated a critical role for nitric oxide synthase (NOS) activation in modulating MeHg neurotoxicity in cerebellar and cortical regions. The aim of the present study is to describe the effects of MeHg on cell viability and NOS activation in chick retinal cell cultures. For this purpose, primary cultures were prepared from 7-day-old chick embryos: retinas were aseptically dissected and dissociated and cells were grown at 37ºC for 7-8 days. Cultures were exposed to MeHg (10 µM, 100 µM, and 1 mM) for 2, 4, and 6 h. Cell viability was measured by MTT method and NOS activity by monitoring the conversion of L-[H³]-arginine to L-[H³]-citrulline. The incubation of cultured retina cells with 10 and 100 µM MeHg promoted an increase of NOS activity compared to control (P < 0.05). Maximum values (P < 0.05) were reached after 4 h of MeHg incubation: increases of 81.6 ± 5.3 and 91.3 ± 3.7%, respectively (data are reported as mean ± SEM for 4 replicates). MeHg also promoted a concentration- and time-dependent decrease in cell viability, with the highest toxicity (a reduction of about 80% in cell viability) being observed at the concentration of 1 mM and after 4-6 h of incubation. The present study demonstrates for the first time the modulation of MeHg neurotoxicity in retinal cells by the nitrergic system.

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel&#8482;-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

The use of polymerase chain reaction for early diagnosis of tuberculosis in Mycobacterium tuberculosis culture

Early diagnosis plays a vital role in controlling tuberculosis. The conventional methodology is slow, with results taking several weeks, in addition to having low sensitivity, especially in clinical paucibacillary samples. The objective of this study was to evaluate the use of polymerase chain reaction (PCR) on solid medium culture for a rapid diagnosis of tuberculosis, mainly in cases of negative sputum smears. Forty sputum samples were collected from inpatients with tuberculosis treated for less than 2 days. Bacilloscopy, PCR for sputum, culture on Löwestein-Jensen (LJ) solid medium, and daily PCR from culture were performed on each sample. DNA extracted from the BCG vaccine, which contains attenuated bacillus Calmette-Guérin, was used as the positive control. Smear microscopy showed 68.6% sensitivity, 80% specificity, 96% positive predictive value, and 26.7% negative predictive value, with culture on LJ medium as the gold standard. Culture at day 28 showed 74.3% sensitivity and 100% specificity. PCR of DNA extracted from sputum amplified a 1027-bp fragment of the 16s RNA gene, showing 22.9% sensitivity and 60% specificity. PCR performed with DNA extracted from daily culture showed that, from the 17th to the 40th day, the sensitivity (85.7%) and specificity (60%) were constant. We conclude that a 17-day culture is a good choice for rapid diagnosis and to interfere with the transmission chain of tuberculosis.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.