Influência de fatores ambientais sobre o crescimento in vitro de Hypnea musciformis (Wulfen) Lamouroux (Rhodophyta)

Hypnea musciformis (Wulfen) Lamourox (Hypneaceae, Gigartinales) é uma fonte importante para o Brasil de um agente ficocolóide geleificante (carragenano) usado na indústria. O objetivo deste estudo foi o de investigar a influência da temperatura (20 e 25 ºC), do enriquecimento água do mar com a solução de Provasoli e aeração sobre o desenvolvimento desta espécie in vitro. Os talos de H. musciformis foram coletados na Prainha, Arraial do Cabo (RJ) em duas diferentes épocas do ano de 2000 (abril e julho). No laboratório, fragmentos apicais pesando 0,2 g (15 mm de comprimento) foram excisados do talo e preparados para as incubações em diferentes combinações de fatores. Em ordem decrescente, a análise de variância multifatorial mostrou que os fatores de maior influência sobre o crescimento dos fragmentos apicais dos talos de H. musciformis foram: enriquecimento, aeração, a temperatura e o período do ano. Todos os fragmentos tiveram aumento de biomassa mostrando plasticidade fenotípica durante o período experimental. Os melhores resultados foram obtidos em culturas com meio de enriquecimento, com aeração e a 25 ºC (biomassa de 17 g e taxa específica de crescimento de 20,79 %.dia-1). Por outro lado, H. musciformis não respondeu bem em culturas não enriquecidas, ausência de aeração e a 25 ºC (biomassa de 0,7 e taxa de crescimento de 0,07 %.dia-1). As plantas cultivadas nas duas épocas do ano não demonstraram diferenças significativas. Concluí-se que a partir dos fragmentos apicais de H. musciformis cultivados, obteve-se bons resultados que fornecerão uma base para futuras aplicações.

In vitro somatic embryogenesis and adventitious root initiation have a common origin in eggplant (Solanum melongena L.)

Somatic embryogenesis was induced from cotyledon explants of eggplant cultured on MS medium supplemented with 54 µM NAA. Anatomical analysis of somatic embryo initiation and development was performed during the first four weeks. Proembryo formation was observed after the second day of culture, directly from perivascular cells or via pro-embryogenic masses derived from indeterminate meristematic masses (IMMs) originated in the vascular tissue. Those IMMs also gave rise to root primordia after 10 days of culture. The origin of embryos is discussed as well as the similarities between somatic embryogenesis and adventitious root formation.

Ultrastructural analysis of in vitro direct and indirect organogenesis

This is a comparative study of the ultrastructural characteristics of the cells involved in the organogenesis in vitro of Bauhinia forficata Link (indirect system) and Glycine max (L.) Merrill (direct system). B. forficata calli after 30 days culture and G. max meristemoids after 10 days culture were prepared for ultrastructural analysis using conventional methods. Concentrically arranged rough endoplasmic reticulum (RER) and plastids containing starch grains were seen during G. max and B. forficata organogenesis. The amitotic process, the presence of plastids around the nucleus and nuclear envelope with conspicuous pores were found in B. forficata.

In vitro and in vivo studies demonstrate non-mutagenicity of the herbicide metolachlor

The herbicide metolachlor was evaluated for genotoxic potential. Metolachlor did not induce micronuclei in mice, however at 40 mg/kg it significantly decreased the percentage of polychromatic erythrocytes, which is a cytotoxic effect. Metolachlor did not induce chromosomal aberrations in human lymphocytes in vitro, but 2.0 mug/ml culture medium resulted in cytotoxicity, decreasing the mitotic index significantly. The indirect exposure test was carried out by adding plasma from metolachlor-pretreated rats to the human lymphocyte cultures. There was no indication of clastogenicity by metolachlor metabolites. On the other hand, plasma of cyclophosphamide-pretreated rats had a significant clastogenic effect

Veratridine increases the survival of retinal ganglion cells in vitro

Neuronal cell death is an important phenomenon involving many biochemical pathways. This degenerative event has been studied to understand how the cells activate the mechanisms that lead to self-destruction. Target cells and afferent cells play a relevant role in the regulation of natural cell death. We studied the effect of veratridine (1.5, 3.0, 4.5 and 6.0 µM) on the survival of neonatal rat retinal ganglion cells in vitro. Veratridine (3.0 µM), a well-known depolarizing agent that opens the Na+ channel, promoted a two-fold increase in the survival of retinal ganglion cells kept in culture for 48 h. This effect was dose-dependent and was blocked by 1.0 µM tetrodotoxin (a classical voltage-dependent Na+ channel blocker) and 30.0 µM flunarizine (a Na+ and Ca2+ channel blocker). These results indicate that electrical activity is also important for the maintenance of retinal ganglion cell survival in vitro

Effects of sciatic-conditioned medium on neonatal rat retinal cells in vitro

Schwann cells produce and release trophic factors that induce the regeneration and survival of neurons following lesions in the peripheral nerves. In the present study we examined the in vitro ability of developing rat retinal cells to respond to factors released from fragments of sciatic nerve. Treatment of neonatal rat retinal cells with sciatic-conditioned medium (SCM) for 48 h induced an increase of 92.5 ± 8.8% (N = 7 for each group) in the amount of total protein. SCM increased cell adhesion, neuronal survival and glial cell proliferation as evaluated by morphological criteria. This effect was completely blocked by 2.5 µM chelerythrine chloride, an inhibitor of protein kinase C (PKC). These data indicate that PKC activation is involved in the effect of SCM on retinal cells and demonstrate that fragments of sciatic nerve release trophic factors having a remarkable effect on neonatal rat retinal cells in culture.

A biolistic process for in vitro gene transfer into chicken embryos

Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100%, survival rate 25% and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters.

Insulin impairs the maturation of chondrocytes in vitro

The precise nature of hormones and growth factors directly responsible for cartilage maturation is still largely unclear. Since longitudinal bone growth occurs through endochondral bone formation, excess or deficiency of most hormones and growth factors strongly influences final adult height. The structure and composition of the cartilaginous extracellular matrix have a critical role in regulating the behavior of growth plate chondrocytes. Therefore, the maintenance of the three-dimensional cell-matrix interaction is necessary to study the influence of individual signaling molecules on chondrogenesis, cartilage maturation and calcification. To investigate the effects of insulin on both proliferation and induction of hypertrophy in chondrocytes in vitro we used high-density micromass cultures of chick embryonic limb mesenchymal cells. Culture medium was supplemented with 1% FCS + 60 ng/ml (0.01 µM) insulin and cultures were harvested at regular time points for later analysis. Proliferating cell nuclear antigen immunoreactivity was widely detected in insulin-treated cultures and persisted until day 21 and [³H]-thymidine uptake was highest on day 14. While apoptosis increased in control cultures as a function of culture time, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-labeled cells were markedly reduced in the presence of insulin. Type II collagen production, alkaline phosphatase activity and cell size were also lower in insulin-treated cultures. Our results indicate that under the influence of 60 ng/ml insulin, chick chondrocytes maintain their proliferative potential but do not become hypertrophic, suggesting that insulin can affect the regulation of chondrocyte maturation and hypertrophy, possibly through an antiapoptotic effect.

Maturation of pig oocytes in vitro in a medium with pyruvate

The aim of in vitro maturation oocyte systems is to produce oocytes of comparable quality to those derived in vivo. The present study was designed to examine the surface morphological changes of the cumulus-oocyte complex (COC) and nuclear maturation in a culture system containing pyruvate. Ovaries were obtained from a slaughterhouseand transported to the laboratory within 2 h at 35-39ºC,and rinsed three times in 0.9% NaCl. The COCs were harvested from the ovaries and in vitro maturation was evaluated in San Marcos (SM) medium, a chemically defined culture system containing 22.3 mM sodium pyruvate. Oocytes were cultured in SM, SM + porcine follicular fluid (pFF) and in SM + pFF + gonadotropins (eCG and hCG) for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Oocytes matured in SM (40.9%) and SM + pFF (42.9%) showed moderate cumulus expansion, whereas oocytes matured in SM + pFF + gonadotropins (54.6%) showed high cumulus expansion. The maturation rate of cultured oocytes, measured in function of the presence of the polar corpuscle, did not differ significantly between SM (40.9 ± 3.6%) and SM + pFF (42.9 ± 3.7%). These results indicate that pig oocytes can be successfully matured in a chemically definedmedium and suggest a possible bifunctional role of pyruvate as an energy substrate and as an antioxidant protecting oocytes against the stress of the in vitro environment.

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.

Trypanosoma cruzi infection induces up-regulation of cardiac muscarinic acetylcholine receptors in vivo and in vitro

The pathogenesis of chagasic cardiomyopathy is not completely understood, but it has been correlated with parasympathetic denervation (neurogenic theory) and inflammatory activity (immunogenic theory) that could affect heart muscarinic acetylcholine receptor (mAChR) expression. In order to further understand whether neurogenic and/or immunogenic alterations are related to changes in mAChR expression, we studied two models of Trypanosoma cruzi infection: 1) in 3-week-old male Sprague Dawley rats chronically infected with T. cruzi and 2) isolated primary cardiomyocytes co-cultured with T. cruzi and peripheral blood mononuclear cells (PBMC). Using [³H]-quinuclidinylbenzilate ([³H]-QNB) binding assays, we evaluated mAChR expression in homogenates from selected cardiac regions, PBMC, and cultured cardiomyocytes. We also determined in vitro protein expression and pro-inflammatory cytokine expression in serum and cell culture medium by ELISA. Our results showed that: 1) mAChR were significantly (P < 0.05) up-regulated in right ventricular myocardium (means ± SEM; control: 58.69 ± 5.54, N = 29; Chagas: 72.29 ± 5.79 fmol/mg, N = 34) and PBMC (control: 12.88 ± 2.45, N = 18; Chagas: 20.22 ± 1.82 fmol/mg, N = 19), as well as in cardiomyocyte transmembranes cultured with either PBMC/T. cruzi co-cultures (control: 24.33 ± 3.83; Chagas: 43.62 ± 5.08 fmol/mg, N = 7 for both) or their conditioned medium (control: 37.84 ± 3.84, N = 4; Chagas: 54.38 ± 6.28 fmol/mg, N = 20); 2) [³H]-leucine uptake was increased in cardiomyocytes co-cultured with PBMC/T. cruzi-conditioned medium (Chagas: 21,030 ± 2321; control 10,940 ± 2385 dpm, N = 7 for both; P < 0.05); 3) plasma IL-6 was increased in chagasic rats, IL-1β, was increased in both plasma of chagasic rats and in the culture medium, and TNF-α level was decreased in the culture medium. In conclusion, our results suggest that cytokines are involved in the up-regulation of mAChR in chronic Chagas disease.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (α-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), α-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with α-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

In vitro studies of multiwalled carbon nanotube/ultrahigh molecular weight polyethylene nanocomposites with osteoblast-like MG63 cells

Carbon nanotubes are highly versatile materials; new applications using them are continuously being developed. Special attention is being dedicated to the possible use of multiwalled carbon nanotubes in biomaterials contacting with bone. However, carbon nanotubes are also controversial in regards to effects exerted on living organisms. Carbon nanotubes can be used to improve the tribological properties of polymer/composite materials. Ultrahigh molecular weight polyethylene (UHMWPE) is a polymer widely used in orthopedic applications that imply wear and particle generation. We describe here the response of human osteoblast-like MG63 cells after 6 days of culture in contact with artificially generated particles from both UHMWPE polymer and multiwalled carbon nanotubes (MWCNT)/UHMWPE nanocomposites. This novel composite has superior wear behavior, having thus the potential to reduce the number of revision hip arthroplasty surgeries required by wear failure of acetabular cups and diminish particle-induced osteolysis. The results of an in vitro study of viability and proliferation and interleukin-6 (IL-6) production suggest good cytocompatibility, similar to that of conventional UHMWPE (WST-1 assay results are reported as percentage of control ± SD: UHMWPE = 96.19 ± 7.92, MWCNT/UHMWPE = 97.92 ± 8.29%; total protein: control = 139.73 ± 10.78, UHMWPE = 137.07 ± 6.17, MWCNT/UHMWPE = 163.29 ± 11.81 µg/mL; IL-6: control = 90.93 ± 10.30, UHMWPE = 92.52 ± 11.02, MWCNT/UHMWPE = 108.99 ± 9.90 pg/mL). Standard cell culture conditions were considered as control. These results, especially the absence of significant elevation in the osteolysis inductor IL-6 values, reinforce the potential of this superior wear-resistant composite for future orthopedic applications, when compared to traditional UHMWPE.

Supraorganized collagen enhances Schwann cell reactivity and organization in vitro

We investigated the reactivity and expression of basal lamina collagen by Schwann cells (SCs) cultivated on a supraorganized bovine-derived collagen substrate. SC cultures were obtained from sciatic nerves of neonatal Sprague-Dawley rats and seeded on 24-well culture plates containing collagen substrate. The homogeneity of the cultures was evaluated with an SC marker antibody (anti-S-100). After 1 week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, S-100 and p75NTR (pan neurotrophin receptor) and for scanning electron microscopy (SEM). Positive labeling with antibodies to the cited molecules was observed, indicating that the collagen substrate stimulates SC alignment and adhesion (collagen IV labeling - organized collagen substrate: 706.33 ± 370.86, non-organized collagen substrate: 744.00 ± 262.09; S-100 labeling - organized collagen: 3809.00 ± 120.28, non-organized collagen: 3026.00 ± 144.63, P < 0.05) and reactivity (p75NTR labeling - organized collagen: 2156.33 ± 561.78, non-organized collagen: 1424.00 ± 405.90, P < 0.05; means ± standard error of the mean in absorbance units). Cell alignment and adhesion to the substrate were confirmed by SEM analysis. The present results indicate that the collagen substrate with an aligned suprastructure, as seen by polarized light microscopy, provides an adequate scaffold for SCs, which in turn may increase the efficiency of the nerve regenerative process after in vivo repair.

Modulation of murine blastocyst hatching in vitro by glutamine and tryptophan

Enrichment of culture media with amino acids improves embryo development. However, little is known about the specific action of each amino acid during embryogenesis. The present study was undertaken to examine the effect of L-glutamine (Gln) and tryptophan (Trp) on mouse embryo hatching, expansion and viability in vitro. Blastocysts were collected from 6- to 8-week-old female BALB/c mice (N = 30) and cultured in M2 medium containing either 0.125, 0.25 or 0.5 mM Trp, 1 mM Gln, or M2 alone. Gln significantly increased (100%; P < 0.05) blastocyst hatching at 24 h compared to M2 alone or Trp; moreover, Trp inhibited blastocyst hatching when compared to M2 alone (P < 0.05) at 72 h. In contrast, the percentage of embryos reaching the state of expanded blastocyst at 48 h was significantly higher in medium with 1 mM Gln (66.6%; P < 0.05) or with 0.125 mM Trp (61.1%; P < 0.05). Unexpectedly, Trp increased the percentage of degenerated blastocysts after 48 h (67.7%; P < 0.05), while Gln preserved blastocyst viability. These results suggest that Gln may enhance blastocyst hatching, expansion and viability in vitro.