Label-free cell separation and sorting in microfluidic systems

Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible

The impact of immediate breast reconstruction on the time to delivery of adjuvant therapy: the iBRA-2 study

Background: Immediate breast reconstruction (IBR) is routinely offered to improve quality-of-life for women requiring mastectomy, but there are concerns that more complex surgery may delay adjuvant oncological treatments and compromise long-term outcomes. High-quality evidence is lacking. The iBRA-2 study aimed to investigate the impact of IBR on time to adjuvant therapy. Methods: Consecutive women undergoing mastectomy ± IBR for breast cancer July–December, 2016 were included. Patient demographics, operative, oncological and complication data were collected. Time from last definitive cancer surgery to first adjuvant treatment for patients undergoing mastectomy ± IBR were compared and risk factors associated with delays explored. Results: A total of 2540 patients were recruited from 76 centres; 1008 (39.7%) underwent IBR (implant-only [n = 675, 26.6%]; pedicled flaps [n = 105,4.1%] and free-flaps [n = 228, 8.9%]). Complications requiring re-admission or re-operation were significantly more common in patients undergoing IBR than those receiving mastectomy. Adjuvant chemotherapy or radiotherapy was required by 1235 (48.6%) patients. No clinically significant differences were seen in time to adjuvant therapy between patient groups but major complications irrespective of surgery received were significantly associated with treatment delays. Conclusions: IBR does not result in clinically significant delays to adjuvant therapy, but post-operative complications are associated with treatment delays. Strategies to minimise complications, including careful patient selection, are required to improve outcomes for patients

Toll-like receptor-4 drives pro-inflammatory cytokine response & tissue degradation in human bacterial keratitis

Purpose: : Toll-like Receptor-4 (TLR4) is a key component of the innate immune response during bacterial infections. Pathways and downstream effectors relating to TLR signalling in human bacterial keratitis (BK) remain unknown. By activating the TLR4 signalling cascade with bacterial lipoploysaccharide (LPS), we investigated whether TLR4 influenced matrix metalloproteases (MMP-2, MMP-9) and cytokine expression in diseased human primary corneal fibroblast (CF) cells are altered.Methods: : Human primary CF cells from patients with severe corneal ulceration from patients with gram negative bacterial keratitis were grown ex vivo and cultured in conjunction with healthy controls. CF cells were treated with exogenous LPS derived from Pseudomonas Aeruginosa.Results: : TLR4, MMP-2 and MMP-9 were constitutively expressed in both ulcerated and control CF cells. Diseased CF cells showed greater responsiveness to LPS stimulation. TLR4 and MMP-9 expression was dose-dependently increased by LPS. MMP-2 expression was not affected by LPS. Analysis on cytokine expression revealed that IL-2, IL-8, IL-10, IL-12p70, GM-CSF, IFNγ and TNF-α expression increased following LPS treatment but only in the diseased cells.Conclusions: : TLR4 activation with LPS increases TLR4, MMP-9 and cytokine expression in CF cells cultured from human BK patients. Over expression of these products may provide a local mechanism to eradicate bacterial infection but also contribute to corneal ulceration and perforation. This is the first description of the effect of TLR4 activation in human bacterial keratitis

Lipopolysaccharide regulation of toll-like receptor-4 and matrix metalloprotease-9 in human primary corneal fibroblast cells

Purpose: Toll-like Receptor 4 (TLR4) is a key component of the innate immune response related to microbial keratitis (MK). Pathways and downstream effectors relating to TLR signalling remain unknown in human bacterial MK. To this effect, by activating the TLR4 signalling cascade with LPS we investigated whether TLR4, matrix metalloproteases (MMP)-2, MMP-9 and cytokine expression in diseased human primary corneal fibroblast (CF) cells are altered. Methods: Human primary CF cells from patients with severe corneal ulceration were cultured in conjunction with healthy control CF cells and treated with lipopolysaccharides (LPS) derived from Pseudomonas aeruginosa. Results: TLR4, MMP-2 and MMP-9 were constitutively expressed in both ulcerated and control CF cells. Diseased CF cells showed greater responsiveness to LPS stimulation. TLR4 and MMP-9 expression was dose-dependently increased by LPS. MMP-2 expression was not affected by LPS. Analysis on cytokine expression revealed that IL-2, IL-8, IL-10, IL-12p70, GM-CSF, IFN? and TNF? expression increased following LPS treatment but only in the diseased cells. Conclusions: TLR4 activation with LPS increases TLR4, MMP-9 and cytokine expression in CF cells cultured from human MK patients. Over expression of these products may provide a local mechanism to eradicate bacterial infection but may also aid corneal ulceration and perforation