Simultaneous determination of irinotecan (CPT-11) and SN-38 in tissue culture media and cancer cells by high performance liquid chromatography: Application to cellular metabolism and accumulation studies

A simple and sensitive HPLC method was developed to simultaneously determine CPT-11 and its major metabolite SN-38 in culture media and cell lysates. Camptothecin (CPT) was used as internal standard (I.S.). Compounds were eluted with acetonitrile–50 mM disodium hydrogen phosphate buffer containing 10 mM sodium 1-heptane-sulfonate, with the pH adjusted to 3.0 using 85% (w/v) orthophosphoric acid (27/73, v/v) by a Hyperclon ODS (C18) column (200 mm × 4.6 mm i.d.), with detection at excitation and emission wavelengths of 380 and 540 nm, respectively. The average extraction efficiencies were 96.9–108.3% for CPT-11 in culture media and 94.3–107.2% for CPT-11 in cell lysates; and 87.7–106.8% for SN-38 in culture media and 90.1–105.6% for SN-38 in cell lysates. Within- and between-day precision and accuracy varied from 0.1 to 10.3%. The limit of quantitation (precision and accuracy <20%) was 5.0 and 2.0 ng/ml for CPT-11 and 1.0 and 0.5 ng/ml for SN-38 in culture media and cell lysates, respectively. This method was successfully applied to quantitate the cellular accumulation and metabolism of CPT-11 and SN-38 in H4-II-E, a rat hepatoma cell line.

Development of a novel pulsatile bioreactor for tissue culture

The construction of tissue-engineered parts such as heart valves and arteries requires more than just the seeding of cells onto a biocompatible/biodegradable polymeric scaffold. It is essential that the functionality and mechanical integrity of the cell-seeded scaffold be investigated in vitro prior to in vivo implantation. The correct hemodynamic conditioning would lead to the development of tissues with enhanced mechanical strength and cell viability. Therefore, a bioreactor that can simulate physiological conditions would play an important role in the preparation of tissue-engineered constructs. In this article, we present and discuss the design concepts and criteria, as well as the development, of a multifunctional bioreactor for tissue culture in vitro. The system developed is compact and easily housed in an incubator to maintain sterility of the construct. Moreover, the proposed bioreactor, in addition to mimicking in vivo conditions, is highly flexible, allowing different types of constructs to be exposed to various physiological flow conditions. Initial verification of the hemodynamic parameters using Laser doppler anemometry indicated that the bioreactor performed well and produced the correct physiological conditions.

Three-dimensional tissue scaffolds from interbonded poly(e-caprolactone) fibrous matrices with controlled porosity

In this article, we report on the preparation and cell culture performance of a novel fibrous matrix that has an interbonded fiber architecture, excellent pore interconnectivity, and controlled pore size and porosity. The fibrous matrices were prepared by combining melt-bonding of short synthetic fibers with a template leaching technique. The microcomputed tomography and scanning electron microscopy imaging verified that the fibers in the matrix were highly bonded, forming unique isotropic pore architectures. The average pore size and porosity of the fibrous matrices were controlled by the fiber/template ratio. The matrices having the average pore size of 120, 207, 813, and 994 mm, with the respective porosity of 73%, 88%, 96%, and 97%, were investigated. The applicability of the matrix as a three-dimensional (3D) tissue scaffold for cell culture was demonstrated with two cell lines, rat skin fibroblast and Chinese hamster ovary, and the influences of the matrix porosity and surface area on the cell culture performance were examined. Both cell lines grew successfully in the matrices, but they showed different preferences in pore size and porosity. Compared with two-dimensional tissue culture plates, the cell number on 3D fibrous matrices was increased by 97.27% for the Chinese hamster ovary cells and 49.46% for the fibroblasts after 21 days of culture. The fibroblasts in the matrices not only grew along the fiber surface but also bridged among the fibers, which was much different from those on two-dimensional scaffolds. Such an interbonded fibrous matrix may be useful for developing new fiber-based 3D tissue scaffolds for various cell culture applications.

Characterization of haemolytic phospholipase A2 activity in clinical isolates of Campylobacter concisus

A membrane-bound, haemolytic phospholipase A2 (PLA2) activity was detected in clinical strains of Campylobacter concisus isolated from children with gastroenteritis. The clinical strains were assigned into two molecular groups (genomospecies) based on PCR amplification of their 23S rDNA. This calcium-dependent, heat-stable, haemolytic PLA2 activity was detected in strains from both genomospecies. A crude haemolysin extract (CHE) was initially prepared from cellular outer-membrane proteins of these isolates and was further fractionated by ultrafiltration. The haemolytic activity of the extracted fraction (R30) was retained by ultrafiltration using a 30 kDa molecular mass cut-off filter, and was designated haemolysin extract (HE). Both CHE and HE had PLA2 activity and caused stable vacuolating and cytolytic effects on Chinese hamster ovary cells in tissue culture. Primers for the conserved region of pldA gene (phospholipase A gene) from Campylobacter coli amplified a gene region of 460 bp in all tested isolates, confirming the presence of a homologous PLA gene sequence in C. concisus. The detection of haemolytic PLA₂ activity in C. concisus indicates the presence of a potential virulence factor in this species and supports the hypothesis that C. concisus is a possible opportunistic pathogen.

Virulence determinants of infectious bursal disease virus

The very virulent (vv) pathotype of infectious bursal disease virus (IBDV) has spread rapidly throughout Europe, Asia, and the Middle East. Although Australia is currently unaffected, there remains the potential for incursion of an exotic isolate. The aim of this study was to identify putative virulence determinants of IBDV to facilitate the development of improved diagnostic assays for detection and characterisation of vvIBDV isolates. Sequencing of Indonesian vvIBDV Tasik94 revealed a unique substitution [ A�¨S222] in the hypervariable region (HVR) of viral protein (VP) VP2, which did not appear to impinge on virulence or antigenicity. Phylogenetic analyses indicated that Tasik94 was closely related to Asian and European vvIBDV strains. Extensive alignment of deduced protein sequences across the HVR of VP2 identified residuesI242 I256 and I294 as putative markers of the vv phcnotype. Comparison of the pathology induced by mildly-virulent Australian IBDV 002/73 and Indonesian vvIBDV Tasik94, revealed that histological lesions in the spleen, thymus and bone marrow were restricted to Tasik94-infected birds, suggesting the enhanced pathogenicity of vvIBDV might be attributed to replication in non-bursal lymphoid organs. The biological significance of the VP2 HVR in virulence was assessed using recombinant viruses generated by reverse genetics. Both genomic segments of Australian IBDV 002/73, and recombinant segment A constructs in which the HVR of 002/73 was replaced with the corresponding region of either tissue culture-adapted virus or vvIBDV (Tasik94), were cloned behind T7 RNA polymerase promoter sequences. In vitro transcription/translation of each construct resulted in expression of viral proteins. Co-transfection of synthetic RNA transcripts initiated replication of both tissue culture-adapted parental and recombinant viruses, however attempts to rescue non-adapted viruses in specific-pathogen-free (SPF) chickens were unsuccessful. Nucleotide sequence variation in the HVR of VP2 was exploited for the development of a new diagnostic assay to rapidly detect exotic IBDV isolates, including vvIBDV, using reverse transcription polymerase chain reaction (RT-PCR) amplification and Bmrl restriction enzyme digestion. The assay was capable of differentiating between endemic and exotic IBDV in 96% of 105 isolates sequenced to date.

Visualization of HIV-1 interactions with penile and foreskin epithelia: clues for female-to-male HIV transmission

To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/- 0.0154 and 0.0171 +/- 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/- 0.0079 virions/image) than glans tissue (0.0167 +/- 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/- 0.0188 vs. 0.0151 +/- 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/- 3.908 vs. 12.466 +/- 2.985 μm). Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites for HIV transmission in uncircumcised men.

MicroRNA expression patterns in post-natal mouse skeletal muscle development

BACKGROUND: MiRNAs are essential regulators of skeletal muscle development and homeostasis. To date, the role and regulation of miRNAs in myogenesis have been mostly studied in tissue culture and during embryogenesis. However, little information relating to miRNA regulation during early post-natal skeletal muscle growth in mammals is available. Using a high-throughput miRNA qPCR-based array, followed by stringent statistical and bioinformatics analysis, we describe the expression pattern and putative role of 768 miRNAs in the quadriceps muscle of mice aged 2 days, 2 weeks, 4 weeks and 12 weeks.

RESULTS: Forty-six percent of all measured miRNAs were expressed in mouse quadriceps muscle during the first 12 weeks of life. We report unprecedented changes in miRNA expression levels over time. The expression of a majority of miRNAs significantly decreased with post-natal muscle maturation in vivo. MiRNA clustering identified 2 subsets of miRNAs that are potentially involved in cell proliferation and differentiation, mainly via the regulation of non-muscle specific targets.

CONCLUSION: Collective miRNA expression in mouse quadriceps muscle is subjected to substantial levels of regulation during the first 12 weeks of age. This study identified a new suite of highly conserved miRNAs that are predicted to influence early muscle development. As such it provides novel knowledge pertaining to post-natal myogenesis and muscle regeneration in mammals.

Ti6Ta4Sn alloy and subsequent scaffolding for bone tissue engineering

Porous titanium (Ti) and titanium alloys are promising scaffold biomaterials for bone tissue engineering, because they have the potential to provide new bone tissue ingrowth abilities and low elastic modulus to match that of
natural bone. In the present study, a new highly porous Ti6Ta4Sn alloy scaffold with the addition of biocompatible alloying elements (tantalum (Ta) and tin (Sn)) was prepared using a space-holder sintering method. The
strength of the Ti6Ta4Sn scaffold with a porosity of 75% was found to be significantly higher than that of a pure Ti scaffold with the same porosity. The elastic modulus of the porous alloy can be customized to match that of
human bone by adjusting its porosity. In addition, the porous Ti6Ta4Sn alloy exhibited an interconnected porous structure, which enabled the ingrowth of new bone tissues. Cell culture results revealed that human SaOS₂
osteoblast-like cells grew and spread well on the surfaces of the solid alloy, and throughout the porous scaffold. The surface roughness of the alloy showed a significant effect on the cell behavior, and the optimum surface
roughness range for the adhesion of the SaOS2 cell on the alloy was 0.15 to 0.35 mm. The present study illustrated the feasibility of using the porous Ti6Ta4Sn alloy scaffold as an orthopedic implant material with a special
emphasis on its excellent biomechanical properties and in vitro biocompatibility with a high preference by osteoblast-like cells.

Biomimetic modification of porous TiNbZr alloy scaffold for bone tissue engineering

Porous titanium (Ti) and Ti alloys are important scaffold materials for bone tissue engineering. In the present study, a new type of porous Ti alloy scaffold with biocompatible alloying elements, that is, niobium (Nb) and zirconium (Zr), was prepared by a space-holder sintering method. This porous TiNbZr scaffold with a porosity of 69% exhibits a mechanical strength of 67MPa and an elastic modulus of 3.9GPa, resembling the mechanical properties of cortical bone. To improve the osteoconductivity, a calcium phosphate (Ca/P) coating was applied to the surface of the scaffold using a biomimetic method. The biocompatibility of the porous TiNbZr alloy scaffold before and after the biomimetic modification was assessed using the SaOS2 osteoblast–like cells. Cell culture results indicated that the porous TiNbZr scaffold is more favorable for cell adhesion and proliferation than its solid counterpart. By applying a Ca/P coating, the cell proliferation rate on the Ca/P-coated scaffold was significantly improved. The results suggest that high-strength porous TiNbZr scaffolds with an appropriate osteoconductive coating could be potentially used for bone tissue engineering application.

Electromagnetic field and other physical methods influencing cell growth in mammal cell culture systems

The growth rate of cultured mammalian cells can be influenced by chemical and physical methods such as electromagnetic fields (EMF), light, temperature and plasma. These physical methods have a number of well documented effects on mammalian cells including modification of gene expression, cell cycle, invasion, motility, cell viability, proliferation, apoptosis and mammosphere numbers. A study of the existing literature confirms that the impact of physical method on mammalian cells depends on the cell type, culture environment, exposure time, frequency, wave shape, and amount of dose. The modification of cell proliferation and apoptosis is necessary for cells products, tissue engineering, and therapy. In this article, we reviewed the impact of four physical methods on the growth rate and viability of cells. Plasma is the best method among fours because we can get desired result ranging from increasing cell proliferation to inducing apoptosis depending on the dose.

Tissue engineered, biomimetic acellular matrices for expansion of hematopoietic progenitors

This thesis discusses a novel strategy for ex vivo expansion of human HSPC in a cell free culture system and it suggests methods to improve the functional properties of stromal cell derived ACMs to support ex-vivo HSPC growth.