Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Simple method for culture of peripheral blood lymphocytes of Testudinidae.

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

Desenvolvimento de embriões in vitro na presença de IGF-1, GH e insulina nos meios de maturação de ovócitos e cultivo embrionário

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Assessment of central venous catheter-associated infections using semi-quantitative or quantitative culture methods

Semiquantitative (Maki) and quantitative (Brun- Buisson) culture techniques were employed in the diagnosis of catheter-related bloodstream infections (CRBSI) in patients who have a short-term central venous catheter (inserted for 30 days). The diagnosis of CRBSI was based on the results of semiquantitative and quantitative culture of material from the removed catheters. Catheter tips (118) from 100 patients were evaluated by both methods. Semiquantitative analysis revealed 34 catheters (28.8%) colonized by ≥15 colonyforming units (cfu), while quantitative cultures (34 catheters, 28.8%) showed the growth of ≥103 cfu/mL. Bacteremia was confirmed in four patients by isolating microorganisms of identical species from both catheters and blood samples. Using the semiquantitative culture technique on short-term central venous catheter tips, we have shown that with a cut-off level of ≥15 cfu, the technique had 100.0% sensitivity, specificity of 68.4%, 25.0% positive predictive value (PPV) and 100.0% negative predictive value (NPV), efficiency of 71.4% and a prevalence of 9.5%. The quantitative method, with a cut-off limit of ≥103 cfu/mL, gave identical values: the sensitivity was 100.0%, specificity 68.4%, positive predictive value (PPV) 25.0%, negative predictive value (NPV) 100.0%, efficiency 71.4% and prevalence 9.5%. We concluded that the semiquantitative and quantitative culture methods, evaluated in parallel, for the first time in Brazil, have similar sensitivity and specificity. Keywords: central venous catheter; semi-quantitative culture; quantitative culture; catheter-related bacteremia.

Efeito do estresse subletal com a utilização de LED no desenvolvimento e na qualidade de embriões bovinos produzidos in vitro

Pós-graduação em Medicina Veterinária - FMVZ

Effects of gaseous atmosphere and antioxidants on the development and cryotolerance of bovine embryos at different periods of in vitro culture

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2 degrees C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows (R). Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 +/- 5.94% and 9.43 +/- 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 +/- 3.37 and 8.67 +/- 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2 degrees C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

The effect of melatonin during in vitro maturation (IVM) on DNA damage of cumulus cells (CCs) from bovine cumulus-oocyte complexes (COCs) and embryo development was evaluated. COCs from abattoir ovaries were cultured in maturation medium (MM) with 0.5 mu g/ml FSH and 5.0 mu g/ml LH (FSH-LH); 10(-9) M melatonin (MEL) or FSH-LH + MEL (FSH-LH-MEL). After 24 h of in vitro maturation, the CCs surrounding the oocyte were subjected to DNA analysis by Comet assay. After in vitro fertilization and in vitro embryo culture, the embryo development rates were evaluated on day 2 post insemination (cleavage) and days 7-8 (blastocyst). The percentage of CCs with no DNA damage was significantly superior in MEL group (37.6 +/- 2.4) than in FSH-LH-MEL (28.0 +/- 2.4) and FSH-LH (17.8 +/- 2.41) groups. Cleavage and blastocysts rates were similar among groups. Melatonin during IVM protects the CCs from DNA damage but this effect did not influence embryo development in vitro. (C) 2010 Elsevier Ltd. All rights reserved.

Axenic cell culture of the seagrass Cymodocea nodosa from cotyledonary tissue

[EN] Plant Tissue Culture, also called “micropropagation”, is the propagation of plants from different tissues (or explants) in a shorter time than conventional propagation, making use of the ability that many plant cells have to regenerate a whole plant (totipotency).There are two alternative mechanisms by which an explant can regenerate an entire plant, namely organogenesis and somatic embryogenesis. Since the last decades, the number of higher terrestrial plants species from which these techniques have been successfully applied has continually increased. However, few attempts have been carried out in marine plants. Previous seagrasses authors have focused their studies on i) vegetative propagation of rhizome fragments as explants in Ruppia maritima, Halophila engelmannii, Cymodocea nodosa and Posidonia oceanica; ii) culture of meristems in Heterozostera tasmanica, C. nodosa or P. oceanica; and iii) culture of germinated seeds on aseptic conditions, in Thalassia testudinum, H. ovalis, P. coriacea, P. oceanica, and H. decipiens. All these studies determine the most adequate culture medium for each species (seawater, nutrients, vitamins, carbon sources, etc...), often supplemented with different plant growth regulators and the necessary conditions for the culture maintenance, such as light and temperature. On the other hand, several studies have previously established protocols for cell or protoplast isolation in the species Zostera marina, Z. muelleri, P. oceanica, and C. nodosa, using shoots collected from natural meadows as original vegetal source, but further cell growth was never accomplished. Due to the absence of somatic embryogenesis or organogenetic studies in seagrasses we wonder: IS THE SUCCESSFUL APPLICATION OF TISSUE CULTURE TECHNIQUES POSSIBLE IN SEAGRASSES?

Porous polyurethane coatings bonded onto surface-modified titanium substrates for the growth of an endothelial cell layer

Cardiovascular diseases refer to the class of diseases that involve the heart or blood vessels (arteries and veins). Examples of medical devices for treating the cardiovascular diseases include ventricular assist devices (VADs), artificial heart valves and stents. Metallic biomaterials such as titanium and its alloy are commonly used for ventricular assist devices. However, titanium and its alloy show unacceptable thrombosis, which represents a major obstacle to be overcome. Polyurethane (PU) polymer has better blood compatibility and has been used widely in cardiovascular devices. Thus one aim of the project was to coat a PU polymer onto a titanium substrate by increasing the surface roughness, and surface functionality. Since the endothelium of a blood vessel has the most ideal non-thrombogenic properties, it was the target of this research project to grow an endothelial cell layer as a biological coating based on the tissue engineering strategy. However, seeding endothelial cells on the smooth PU coating surfaces is problematic due to the quick loss of seeded cells which do not adhere to the PU surface. Thus it was another aim of the project to create a porous PU top layer on the dense PU pre-layer-coated titanium substrate. The method of preparing the porous PU layer was based on the solvent casting/particulate leaching (SCPL) modified with centrifugation. Without the step of centrifugation, the distribution of the salt particles was not uniform within the polymer solution, and the degree of interconnection between the salt particles was not well controlled. Using the centrifugal treatment, the pore distribution became uniform and the pore interconnectivity was improved even at a high polymer solution concentration (20%) as the maximal salt weight was added in the polymer solution. The titanium surfaces were modified by alkli and heat treatment, followed by functionlisation using hydrogen peroxide. A silane coupling agent was coated before the application of the dense PU pre-layer and the porous PU top layer. The ability of the porous top layer to grow and retain the endothelial cells was also assessed through cell culture techniques. The bonding strengths of the PU coatings to the modified titanium substrates were measured and related to the surface morphologies. The outcome of the project is that it has laid a foundation to achieve the strategy of endothelialisation for the blood compatibility of medical devices. This thesis is divided into seven chapters. Chapter 2 describes the current state of the art in the field of surface modification in cardiovascular devices such as ventricular assist devices (VADs). It also analyses the pros and cons of the existing coatings, particularly in the context of this research. The surface coatings for VADs have evolved from early organic/ inorganic (passive) coatings, to bioactive coatings (e.g. biomolecules), and to cell-based coatings. Based on the commercial applications and the potential of the coatings, the relevant review is focused on the following six types of coatings: (1) titanium nitride (TiN) coatings, (2) diamond-like carbon (DLC) coatings, (3) 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer coatings, (4) heparin coatings, (5) textured surfaces, and (6) endothelial cell lining. Chapter 3 reviews the polymer scaffolds and one relevant fabrication method. In tissue engineering, the function of a polymeric material is to provide a 3-dimensional architecture (scaffold) which is typically used to accommodate transplanted cells and to guide their growth and the regeneration of tissue. The success of these systems is dependent on the design of the tissue engineering scaffolds. Chapter 4 describes chemical surface treatments for titanium and titanium alloys to increase the bond strength to polymer by altering the substrate surface, for example, by increasing surface roughness or changing surface chemistry. The nature of the surface treatment prior to bonding is found to be a major factor controlling the bonding strength. By increasing surface roughness, an increase in surface area occurs, which allows the adhesive to flow in and around the irregularities on the surface to form a mechanical bond. Changing surface chemistry also results in the formation of a chemical bond. Chapter 5 shows that bond strengths between titanium and polyurethane could be significantly improved by surface treating the titanium prior to bonding. Alkaline heat treatment and H2O2 treatment were applied to change the surface roughness and the surface chemistry of titanium. Surface treatment increases the bond strength by altering the substrate surface in a number of ways, including increasing the surface roughness and changing the surface chemistry. Chapter 6 deals with the characterization of the polyurethane scaffolds, which were fabricated using an enhanced solvent casting/particulate (salt) leaching (SCPL) method developed for preparing three-dimensional porous scaffolds for cardiac tissue engineering. The enhanced method involves the combination of a conventional SCPL method and a step of centrifugation, with the centrifugation being employed to improve the pore uniformity and interconnectivity of the scaffolds. It is shown that the enhanced SCPL method and a collagen coating resulted in a spatially uniform distribution of cells throughout the collagen-coated PU scaffolds.In Chapter 7, the enhanced SCPL method is used to form porous features on the polyurethane-coated titanium substrate. The cavities anchored the endothelial cells to remain on the blood contacting surfaces. It is shown that the surface porosities created by the enhanced SCPL may be useful in forming a stable endothelial layer upon the blood contacting surface. Chapter 8 finally summarises the entire work performed on the fabrication and analysis of the polymer-Ti bonding, the enhanced SCPL method and the PU microporous surface on the metallic substrate. It then outlines the possibilities for future work and research in this area.

Human preadipocyte proliferation and differentiation

Obesity represents a major health, social and economic burden to many developing and Westernized communities, with the prevalence increasing at a rate exceeding almost all other medical conditions. Despite major recent advances in our understanding of adipose tissue metabolism and dynamics, we still have limited insight into the regulation of adipose tissue mass in humans. Any significant increase in adipose tissue mass requires proliferation and differentiation of precursor cells (preadipocytes) present in the stromo-vascular compartment of adipose tissue. These processes are very complex and an increasing number of growth factors and hormones have been shown to modulate the expression of genes involved in preadipocyte proliferation and differentiation. A number of transcription factors, including the C/EBP family and PP ARy, have been identified as integral to adipose tissue development and preadipocyte differentiation. Together PP ARy and C/EBPa regulate important events in the activation and maintenance of the terminally differentiated phenotype. The ability of PP ARy to increase transcription through its DNA recognition site is dependent on the binding of ligands. This suggests that an endogenous PP ARy ligand may be an important regulator of adipogenesis. Adipose tissue functions as both the major site of energy storage in the body and as an endocrine organ synthesizing and secreting a number of important molecules involved in regulation of energy balance. For optimum functioning therefore, adipose tissue requires extensive vascularization and previous studies have shown that growth of adipose tissue is preceded by development of a microvascular network. This suggests that paracrine interactions between constituent cells in adipose tissue may be involved in both new capillary formation and fat cell growth. To address this hypothesis the work in this project was aimed at (a) further development of a method for inducing preadipocyte differentiation in subcultured human cells; (b) establishing a method for simultaneous isolation and separate culture of both preadipocytes and microvascular endothelial cells from the same adipose tissue biopsies; (c) to determine, using conditioned medium and co-culture techniques, if endothelial cell-derived factors influence the proliferation and/or differentiation of human preadipocytes; and (d) commence characterization of factors that may be responsible for any observed paracrine effects on aspects of human adipogenesis. Major findings of these studies were as follows: (A) Inclusion of either linoleic acid (a long-chain fatty acid reported to be a naturally occurring ligand for PP ARy) or Rosiglitazone (a member of the thiazolidinedione class of insulin-sensitizing drugs and a synthetic PPARy ligand) in differentiation medium had markedly different effects on preadipocyte differentiation. These studies showed that human preadipocytes have the potential to accumulate triacylglycerol irrespective of their stage of biochemical differentiation, and that thiazolidinediones and fatty acids may exert their adipogenic and lipogenic effects via different biochemical pathways. It was concluded that Rosiglitazone is a more potent inducer of human preadipocyte differentiation than linoleic acid. (B) A method for isolation and culture of both endothelial cells and preadipocytes from the same adipose tissue biopsy was developed. Adipose-derived microvascular endothelial cells were found to produce factor/s, which enhance both proliferation and differentiation of human preadipocytes. (C) The adipogenic effects of microvascular endothelial cells can be mimicked by exposure of preadipocytes to members of the Fibroblast Growth Factor family, specifically ~-ECGF and FGF-1. (D) Co-culture of human preadipocytes with endothelial cells or exposure of preadipocytes to either ~-ECGF or FGF-1 were found to 'prime' human preadipocytes, during their proliferative phase of growth, for thiazolidinedione-induced differentiation. (E) FGF -1 was not found to be acting as a ligand for PP ARy in this system. Findings from this project represent a significant step forward in our understanding of factors involved in growth of human adipose tissue and may lead to the development of therapeutic strategies aimed at modifying the process. Such strategies would have potential clinical utility in the treatment of obesity and obesity related disorders such as Type II Diabetes.

Glycosaminoglycan and growth factor mediated murine calvarial cell proliferation

Understanding the complex mechanisms underlying bone remodeling is crucial to the development of novel therapeutics. Glycosaminoglycans (GAGs) localised to the extracellular matrix (ECM) of bone are thought to play a key role in mediating aspects of bone development. The influence of isolated GAGs was studied by utilising in vitro murine calvarial monolayer and organ culture model systems. Addition of GAG preparations extracted from the cell surface of human osteoblasts at high concentrations (5 microg/ml) resulted in decreased proliferation of cells and decreased suture width and number of bone lining cells in calvarial sections. When we investigated potential interactions between the growth factors fibroblast growth factor-2 (FGF2), bone morphogenic protein-2 (BMP2) and transforming growth factor-beta1 (TGFbeta1) and the isolated cell surface GAGs, differences between the two model systems emerged. The cell culture system demonstrated a potentiating role for the isolated GAGs in the inhibition of FGF2 and TGFbeta1 actions. In contrast, the organ culture system demonstrated an enhanced stimulation of TFGbeta1 effects. These results emphasise the role of the ECM in mediating the interactions between GAGs and growth factors during bone development and suggest the GAG preparations contain potent inhibitory or stimulatory components able to mediate growth factor activity.

A tan in a test tube -in vitro models for investigating ultraviolet radiation-induced damage in skin

Presently, global rates of skin cancers induced by ultraviolet radiation (UVR) exposure are on the rise. In view of this, current knowledge gaps in the biology of photocarcinogenesis and skin cancer progression urgently need to be addressed. One factor that has limited skin cancer research has been the need for a reproducible and physiologically-relevant model able to represent the complexity of human skin. This review outlines the main currently-used in vitro models of UVR-induced skin damage. This includes the use of conventional two-dimensional cell culture techniques and the major animal models that have been employed in photobiology and photocarcinogenesis research. Additionally, the progression towards the use of cultured skin explants and tissue-engineered skin constructs, and their utility as models of native skin's responses to UVR are described. The inherent advantages and disadvantages of these in vitro systems are also discussed.

3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling

Mesenchymal stem/stromal cells (MSC) are rapidly becoming a leading candidate for use in tissue regeneration, with first generation of therapies being approved for use in orthopaedic repair applications. Capturing the full potential of MSC will likely require the development of novel in vitro culture techniques and devices. Herein we describe the development of a straightforward surface modification of an existing commercial product to enable the efficient study of three dimensional (3D) human bone marrow-derived MSC osteogenic differentiation. Hundreds of 3D microaggregates, of either 42 or 168 cells each, were cultured in osteogenic induction medium and their differentiation was compared with that occurring in traditional two dimensional (2D) monolayer cultures. Osteogenic gene expression and matrix composition was significantly enhanced in the 3D microaggregate cultures. Additionally, BMP-2 gene expression was significantly up-regulated in 3D cultures at day 3 and 7 by approximately 25- and 30-fold, respectively. The difference in BMP-2 gene expression between 2D and 3D cultures was negligible in the more mature day 14 osteogenic cultures. These data support the notion that BMP-2 autocrine signalling is up-regulated in 3D MSC cultures, enhancing osteogenic differentiation. This study provides both mechanistic insight into MSC differentiation, as well as a platform for the efficient generation of microtissue units for further investigation or use in tissue engineering applications.

The Human Fertilisation and Embryology Act 2008 : restrictions on the creation of “saviour siblings” and the relevance of the harm principle

This paper provides an overview of the regulatory developments in the UK which impact on the use of in vitro fertilization (IVF) and embryo screening techniques for the creation of “saviour siblings.” Prior to the changes implemented under the Human Fertilisation and Embryology Act 2008, this specific use of IVF was not addressed by the legislative framework and regulated only by way of policy issued by the Human Fertilisation and Embryology Authority (HFEA). Following the implementation of the statutory reforms, a number of restrictive conditions are now imposed on the face of the legislation. This paper considers whether there is any justification for restricting access to IVF and pre-implantation tissue typing for the creation of “saviour siblings.” The analysis is undertaken by examining the normative factors that have guided the development of the UK regulatory approach prior to the 2008 legislative reforms. The approach adopted in relation to the “saviour sibling” issue is compared to more general HFEA policy, which has prioritized the notion of reproductive choice and determined that restrictions on access are only justified on the basis of harm considerations.