An improved culture protocol for the differentiation and survival of human promyelocytic leukaemia PLB-985 cell-line into mature neutrophil-like granulocytes

Circulating blood neutrophils are short-lived, lack proliferation capacity and cannot be transfected in vitro to express exogenous genes or proteins. These properties have made the ex vivo genetic manipulation of neutrophils challenging and hindered biochemical and molecular studies investigating the function of specific genes and proteins. Improved methodology for differentiating cell lines into mature neutrophil-like phenotypes, with similar morphological and functional properties to blood neutrophils would, therefore, be an important tool to probe the molecular properties of mature cells. The PLB-985 cell line was cultured in RPMI-1640 medium supplemented foetal calf serum (FCS) and penicillin/streptomycin. For induction of differentiation into neutrophil-like cells, the medium was supplemented with sodium pyruvate, N, N-dimethylformamide (DMF) and all-trans retinoic acid (ATRA), FCS and penicillin/streptomycin. The cytokines G-CSF and GM-CSF were used to enhance differentiation, prolong viability and delay the progression of the differentiated cells into apoptosis. The modified culture protocol and conditions induced PLB-985 cells to differentiate into mature, neutrophil-like granulocytes that resembled the morphology of mature blood neutrophils as evident by acquisition of a multi-lobed nucleus and granulated cytoplasm. These modified culture conditions resulted in enhanced differentiation into neutrophil-like cells and the apoptosis of these differentiated cells was delayed by supplementation with cytokines. This experimental system should be useful for studies probing the function of specific genes and proteins in human neutrophils.

Partial hepatectomy enhances the growth of CC531 rat colorectal cancer cells both in vitro and in vivo

Partial hepatectomy (PHx) is the gold standard for the treatment of colorectal cancer liver metastases. However, after removing a substantial amount of hepatic tissue, growth factors are released to induce liver regeneration, which may promote the proliferation of liver micrometastases or circulating tumour cells still present in the patient. The aim of this study is to assess the effect of PHx on the growth of liver metastases induced by intrasplenic cell inoculation as well as on in vitro proliferation of the same cancer cell line. Liver tumours were induced in 18 WAG/RijHsd male rats, by seeding 250,000 syngeneic colorectal cancer cells (CC531) into the spleen. The left lateral lobe of the liver was mobilized and in half of the animals it was removed to achieve a 40% hepatectomy. Twenty-eight days after tumour induction, the animals were sacrificed and the liver was removed and sliced to assess the relative tumour surface area (RTSA%). CC531 cells were cultured in presence of foetal calf serum, non-hepatectomised (NRS) or hepatectomized rat serum (HRS), and their proliferation rate at 24, 48, and 72 h was measured. RTSA% was significantly higher in animals which had undergone PHx than in the controls (non-hepatectomised) (46.98 ± 8.76% vs. 18.73 ± 5.65%; p < 0.05). Analysing each lobe separately, this difference in favour of hepatectomized animals was relevant and statistically significant in the paramedian and caudate lobes. But in the right lobe the difference was scarce and not significant. In vitro, 2.5% HRS achieved stronger proliferative rates than the control cultures (10% FCS) or their equivalent of NRS. In this experimental model, a parallelism has been shown between the effect of PHx on the growth of colorectal cancer cells in the liver and the effect of the serum on those cells in vitro.

Serum Lowers Bioactivity and Uptake of Synthetic Amorphous Silica by Alveolar Macrophages in a Particle Specific Manner

Various cell types are compromised by synthetic amorphous silica (SAS) if they are exposed to SAS under protein-free conditions in vitro. Addition of serum protein can mitigate most SAS effects, but it is not clear whether this is solely caused by protein corona formation and/or altered particle uptake. Because sensitive and reliable mass spectrometric measurements of SiO2 NP are cumbersome, quantitative uptake studies of SAS at the cellular level are largely missing. In this study, we combined the comparison of SAS effects on alveolar macrophages in the presence and absence of foetal calf serum with mass spectrometric measurement of 28Si in alkaline cell lysates. Effects on the release of lactate dehydrogenase, glucuronidase, TNFα and H2O2 of precipitated (SIPERNAT® 50, SIPERNAT® 160) and fumed SAS (AEROSIL® OX50, AEROSIL® 380 F) were lowered close to control level by foetal calf serum (FCS) added to the medium. Using a quantitative high resolution ICP-MS measurement combined with electron microscopy, we found that FCS reduced the uptake of particle mass by 9.9% (SIPERNAT® 50) up to 83.8% (AEROSIL® OX50). Additionally, larger particle agglomerates were less frequent in cells in the presence of FCS. Plotting values for lactate dehydrogenase (LDH), glucuronidase (GLU) or tumour necrosis factor alpha (TNFα) against the mean cellular dose showed the reduction of bioactivity with a particle sedimentation bias. As a whole, the mitigating effects of FCS on precipitated and fumed SAS on alveolar macrophages are caused by a reduction of bioactivity and by a lowered internalization, and both effects occur in a particle specific manner. The method to quantify nanosized SiO2 in cells is a valuable tool for future in vitro studies.

Novel in vitro assays to detect circulating permeability factor(s) in idiopathic focal segmental glomerulosclerosis

Background Many patients with idiopathic focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation (TX). Although several circulating permeability factors (CPFs) responsible for recurrence have been suggested, there is no consensus. To facilitate CPF identification and predict recurrence after TX, there is a need for robust methods that demonstrate the presence of CPFs. Methods Cultured human podocytes (hPods) and human and mouse glomerular endothelial cells (ciGEnC, mGEnC) were exposed to plasmas of FSGS patients with presumed CPFs, and of (disease) controls. A visual scoring assay and flow cytometry analysis of side scatter were used to measured changes in cellular granularity after exposure to plasma. Results Nine out of 13 active disease plasmas of 10 FSGS patients with presumed CPFs induced granularity in hPod in a dose- and time-dependent manner. Corresponding remission plasmas induced no or less granularity in hPod. Similar results were obtained with ciGEnC and mGEnC, although induced granularity was less compared with hPod. Notably, foetal calf serum, healthy plasma and a remission plasma partially blocked FSGS plasma-induced hPod granularity. Conclusions We developed a novel assay in which active disease, presumably CPF-containing, FSGS plasmas induced granularity in cultured hPod. Our results may indicate the presence of CPF inhibitor(s) in healthy and remission plasma. We suggest the presence of a delicate balance between CPF and a CPF inhibitory factor, which is disturbed in patients with active disease. Our novel assays can be applied in future research to identify CPF and CPF inhibitors, and possibly to predict recurrence after TX.

Mapping the Micro-Abrasion Mechanisms of CoCrMo: Some Thoughts on Varying Ceramic Counterface Diameter on Transition Boundaries In Vitro

The micro-abrasion wear mechanisms for CoCrMo against variable size alumina balls, representing typical artificial femoral head sizes, were investigated over a range of applied loads in foetal calf serum solution. SEM analysis of resulting wear scars displayed two-body and mixed-mode abrasion modes of wear. The wear factor, κ, was found to range between 0.86 and 22.87 (10−6 mm3/Nm). Micro-abrasion mechanism and wastage maps were constructed for the parameter range tested. A dominant two- to three-body abrasion regime was observed with an increasing load and ball diameter. The 28-mm ball diameter displayed the lowest wastage, with an increasing load. Proteins may act to reduce the severity of contact between abrasive particles and bearing surfaces. Wear volumes did not necessarily increase linearly with applied load and ball diameter; therefore, there is a need to develop more accurate models for wear prediction during micro-abrasion conditions. Wear mapping for hip replacements could provide a useful aid for pre-clinical hip wear evaluations and long-term performance.

Comparison of two vitrification methods for cryopreservation of porcine embryos

The aim of this study was to compare two vitrification methods of porcine perihatching blastocysts with regard to the success of transfer of these embryos to the recipients. Expanded, hatching, or hatched blastocysts were recovered post mortem from superovulated donors in 5.5 to 6.0 days after artificial insemination of donor gilts with homospermic doses. In protocol VS I, the embryos in perihatching developmental stage were equilibrated in a culture medium H-MEMD with 10% v/v of glycerol (1.37M solution of glycerol in medium) for 10 min and placed in a vitrification medium for 1.5 min max. (vitrification medium contained 50% v/v 2M sucrose in tridistilled water, 30% v/v of glycerol, and 20% v/v of foetal calf serum &ndash; FCS). Then they were dropped with micropipette and stored in liquid nitrogen vapour. For protocol VS II, we used H-MEMD culture medium supplemented with 20% v/v of FCS, 25% v/v ethylene glycol, and 25% v/v dimethyl sulphoxide (DMSO). Embryos were equilibrated for 10&nbsp;min in a mixture of the vitrification medium and culture medium (1 : 1), and were kept in the vitrification medium for 1.5&nbsp;minutes. Then they were dropped with micropipette and stored in liquid nitrogen vapour. Embryos were thawed by immersing the drop with the embryo in H-MEMD culture medium with 0.8M sucrose for 10 minutes. After thawing and washing in the medium with sucrose, all embryos were washed three times in a fresh medium and prepared for transfer. Recipients were synchronized either using Regumate-feeding followed by treatment with PMSG and HCG (gilts) or using piglet weaning (sows &ndash; 1st and 2nd parity). Recipients showing standing heat at the time of donor insemination were used for laparoscopic and non-surgical ET on day 5.5&ndash;6.0 of the cycle. The fraction of viable embryo vitrified under VS I or VS II protocol was 85% and 80%, compared to 95% in control fresh embryos (P &gt; 0.05). Pregnancy of recipients was 57.3% (5/7), 67.0% (4/6) for VS I or VS II group and 42.7% (10/23) for control (P &lt; 0.001). We can conclude on the basis of our data that both protocols for vitrification yielded similar results and can be used for cryopreservation of porcine embryos. &nbsp;

Parthenogenetic development of rabbit oocytes after electrical stimulation

The aim of this study was to determine the effect of electric pulses on the structural and functional condition of rabbit oocytes. The New Zealand White female rabbits at 3&ndash;5 months of age and at 3&ndash;4 kg body weight served as oocyte donors. Oocytes after flushing from the oviducts were placed between two electrodes in an electroporation chamber which was filled with a dielectric solution. Following a short incubation in B2 medium, oocytes were subjected to an electric pulse released by an electrical pulse generator. Oocytes were then incubated in 500 &micro;l of B2 medium supplemented with 20% foetal calf serum (FCS) at 38&deg;C in an atmosphere of 5% CO₂in air. Oocytes were cultured until the morula/blastocyst stage (approx. 72 h). The experiment was conducted using 430 oocytes obtained post mortem. In vitro cultured oocytes not subjected to an electric pulse were the control. Each group was subdivided into replications according to electric current intensity. The analysis of experimental variants shows that in the first variant all embryos developed to the morula stage but only 10% of them continued to develop to the blastocyst stage. In the second variant we observed that 5&ndash;10% of oocytes developed to the blastocyst stage after treatment with 2.0 and 2.5 kV/cm pulse but in the group of 1.0 kV/cm pulse 35% of oocytes developed only to the 2&ndash;12 b stage. In the third variant only 1 oocyte (5%) continued to develop to the blastocyst stage, but in the fourth variant oocyte development stopped at the morula stage. In the fifth variant, called an &ldquo;extreme&rdquo; one, oocytes stopped to develop at the stage of 2&ndash;12 b (about 25%) and the percentage of degenerated oocytes dramatically increased (about 60%). &nbsp;

The Effects of Hyaluronic Acid, Calcium Hydroxide, and Dentin Adhesive on Rat Odontoblasts and Fibroblasts

The Effects of Hyaluronic Acid, Calcium Hydroxide, and Dentin Adhesive on Rat Odontoblasts and FibroblastsThe aim of this study was to investigate the effects and efficiency of pulp capping preparations based on hyaluronic acid, calcium hydroxide, and dentin adhesive on the pulp tissue of Sprague-Dawley rats. The rats were killed and extracted teeth sectioned transversely through the pulp. The slices were placed in a RPMI 1640 cell culture medium supplemented with 10 % foetal calf serum. During 14 days of cultivation cultures were treated with preparations that contained hyaluronic acid (Gengigel Prof®), and calcium hydroxide (ApexCal®), or with dentin adhesive (Excite®). Cellularity and viability of fibroblasts and odontoblasts was analysed using a haemocytometer. Hyaluronic acid proved most efficient and the least toxic for direct pulp capping. Even though calcium hydroxide and dentin adhesive demonstrated a higher degree of cytotoxicity, their effects were still acceptable in terms of biocompatibility.