Cell sorting in a Petri dish controlled by computer vision.

Fluorescence-activated cell sorting (FACS) applying flow cytometry to separate cells on a molecular basis is a widespread method. We demonstrate that both fluorescent and unlabeled live cells in a Petri dish observed with a microscope can be automatically recognized by computer vision and picked up by a computer-controlled micropipette. This method can be routinely applied as a FACS down to the single cell level with a very high selectivity. Sorting resolution, i.e., the minimum distance between two cells from which one could be selectively removed was 50-70 micrometers. Survival rate with a low number of 3T3 mouse fibroblasts and NE-4C neuroectodermal mouse stem cells was 66 +/- 12% and 88 +/- 16%, respectively. Purity of sorted cultures and rate of survival using NE-4C/NE-GFP-4C co-cultures were 95 +/- 2% and 62 +/- 7%, respectively. Hydrodynamic simulations confirmed the experimental sorting efficiency and a cell damage risk similar to that of normal FACS

Gentamicin supplemented polyvinylidenfluoride mesh materials enhance tissue integration due to a transcriptionally reduced MMP-2 protein expression

<p>Abstract</p> <p>Background</p> <p>A beneficial effect of gentamicin supplemented mesh material on tissue integration is known. To further elucidate the interaction of collagen and MMP-2 in chronic foreign body reaction and to determine the significance of the MMP-2-specific regulatory element (RE-1) that is known to mediate 80% of the MMP-2 promoter activity, the spatial and temporal transcriptional regulation of the MMP-2 gene was analyzed at the cellular level.</p> <p>Methods</p> <p>A PVDF mesh material was surface modified by plasma-induced graft polymerization of acrylic acid (PVDF+PAAc). Three different gentamicin concentrations were bound to the provided active sites of the grafted mesh surfaces (2, 5 and 8 μg/mg). 75 male transgenic MMP-2/LacZ mice harbouring the LacZ reporter gene under control of MMP-2 regulatory sequence -1241/+423, excluding the RE-1 were randomized to five groups. Bilateral of the abdominal midline one of the five different meshes was implanted subcutaneously in each animal. MMP-2 gene transcription (anti-ß-galactosidase staining) and MMP-2 protein expression (anti-MMP-2 staining) were analyzed semiquantitatively by immunohistochemistry 7, 21 and 90 days after mesh implantation. The collagen type I/III ratio was analyzed by cross polarization microscopy to determine the quality of mesh integration.</p> <p>Results</p> <p>The perifilamentary ß-galactosidase expression as well as the collagen type I/III ratio increased up to the 90^thday for all mesh modifications, whereas no significant changes could be observed for MMP-2 protein expression between days 21 and 90. Both the 5 and 8 μg/mg gentamicin group showed significantly reduced levels of ß-galactosidase expression and MMP-2 positive stained cells when compared to the PVDF group on day 7, 21 and 90 respectively (5 μg/mg: p < 0.05 each; 8 μg/mg: p < 0.05 each). Though the type I/III collagen ratio increased over time for all mesh modifications significant differences to the PVDF mesh were only detected for the 8 μg/mg group at all 3 time points (p < 0.05 each).</p> <p>Conclusions</p> <p>Our current data indicate that lack of RE-1 is correlated with increased mesh induced MMP-2-gene expression for coated as well as for non-coated mesh materials. Gentamicin coating reduced MMP-2 transcription and protein expression. For the 8 μg/mg group this effect is associated with an increased type I/III collagen ratio. These findings suggest that gentamicin is beneficial for tissue integration after mesh implantation, which possibly is mediated via RE-1.</p

Microbiome to Brain:Unravelling the Multidirectional Axes of Communication

The gut microbiome plays a crucial role in host physiology. Disruption of its community structure and function can have wide-ranging effects making it critical to understand exactly how the interactive dialogue between the host and its microbiota is regulated to maintain homeostasis. An array of multidirectional signalling molecules is clearly involved in the host-microbiome communication. This interactive signalling not only impacts the gastrointestinal tract, where the majority of microbiota resides, but also extends to affect other host systems including the brain and liver as well as the microbiome itself. Understanding the mechanistic principles of this inter-kingdom signalling is fundamental to unravelling how our supraorganism function to maintain wellbeing, subsequently opening up new avenues for microbiome manipulation to favour desirable mental health outcome

Preparation, in vitro release, in vivo absorption and biocompatibility studies of insulin-loaded microspheres in rabbits

The purpose of this study was to develop a single-dose insulin delivery system based on poly (lactide-co-glycolide) (PLGA) microspheres to provide basal insulin level for a prolonged period. Insulin-loaded PLGA microspheres were prepared by water-in-oil-in-water double emulsion (batch A) and solid-in0oil-in-water emulsion (batch B) methods. Microspheres were characterized for physical characteristics and in vitro release. In vivo absorption of insulin and biocompatibility of insulin-loaded PLGA microspheres were performed in diabetic New Zealand white rabbits. Light and transmission electron microscopy were performed on the skin tissues excised from microspheres injected sites in order to study the biocompatibility. The burst release of insulin was high (47%) from batch B and low (5%) from batch A. Therefore, we mixed microspheres of batch A and B in ratio of 3∶ w/w, which produced desirable in vitro release profile. In vivo absorption study showed that insulin-loaded microspheres provided a serum insulin level of 20–40 μU/ml up to 40 days. Biocompatibility study provided evidence of normal inflammatory and foreign body reactions, which were characterized by the presence of macrophages, fibroblasts and foreign body giant cells. Neither necrosis nor tissue damage was identified. At the end of 12 weeks, no distinct histological differences were observed in comparison to the control tissue samples. In conclusion, insulin-loaded PLGA microspheres controlled the in vivo absorption of insulin to maintain the basal insulin level for longer period and the delivery system was biocompatible