A review on microfluidics manipulation of the extracellular chemical microenvironment and its emerging application to cell analysis.

Spatiotemporal manipulation of extracellular chemical environments with simultaneous monitoring of cellular responses plays an essential role in exploring fundamental biological processes and expands our understanding of underlying mechanisms. Despite the rapid progress and promising successes in manipulation strategies, many challenges remain due to the small size of cells and the rapid diffusion of chemical molecules. Fortunately, emerging microfluidic technology has become a powerful approach for precisely controlling the extracellular chemical microenvironment, which benefits from its integration capacity, automation, and high-throughput capability, as well as its high resolution down to submicron. Here, we summarize recent advances in microfluidics manipulation of the extracellular chemical microenvironment, including the following aspects: i) Spatial manipulation of chemical microenvironments realized by convection flow-, diffusion-, and droplet-based microfluidics, and surface chemical modification; ii) Temporal manipulation of chemical microenvironments enabled by flow switching/shifting, moving/flowing cells across laminar flows, integrated microvalves/pumps, and droplet manipulation; iii) Spatiotemporal manipulation of chemical microenvironments implemented by a coupling strategy and open-space microfluidics; and iv) High-throughput manipulation of chemical microenvironments. Finally, we briefly present typical applications of the above-mentioned technical advances in cell-based analyses including cell migration, cell signaling, cell differentiation, multicellular analysis, and drug screening. We further discuss the future improvement of microfluidics manipulation of extracellular chemical microenvironments to fulfill the needs of biological and biomedical research and applications.

The architecture and spatial organization of the living human body as revealed by intratissular endoscopy - An osteopathic perspective.

This article presents an overview of research conducted by Dr Jean-Claude Guimberteau into the architecture and spatial organization of living matter and the relationship between the cells and the extracellular matrix. His research is discussed in the context of previous and current research into fascial anatomy. Andrew Taylor Still, the founder of Osteopathy, did not have access to modern research and yet his observations are proving to be surprisingly accurate in the light of recent findings. This article sets out to highlight the relevance of his insights from a purely anatomical perspective, and to draw parallels with a new way of thinking about the architecture of the living human body that is slowly emerging. Dr Guimberteau's research shows that a force applied to the surface of the skin is transmitted deep into living tissue via a continuous bodywide multifibrillar network. It also confirms the concept of the body as a dynamic functional unit, as proposed by A.T. Still. Still also proposed that structure and function are interrelated at all levels within the living human body. There is a growing body of research to support this. Intratissular endoscopy has highlighted the importance of the quality of the mobility and adaptability of the network of collagen and elastin fibers that structures the ECM in healthy living tissue. Factors such as abnormal stiffness of collagen fibers in the ECM are thought to have adverse effects on local tissue health.

Multi-Organs-on-Chips: Towards Long-Term Biomedical Investigations.

With advantageous features such as minimizing the cost, time, and sample size requirements, organ-on-a-chip (OOC) systems have garnered enormous interest from researchers for their ability for real-time monitoring of physical parameters by mimicking the in vivo microenvironment and the precise responses of xenobiotics, i.e., drug efficacy and toxicity over conventional two-dimensional (2D) and three-dimensional (3D) cell cultures, as well as animal models. Recent advancements of OOC systems have evidenced the fabrication of 'multi-organ-on-chip' (MOC) models, which connect separated organ chambers together to resemble an ideal pharmacokinetic and pharmacodynamic (PK-PD) model for monitoring the complex interactions between multiple organs and the resultant dynamic responses of multiple organs to pharmaceutical compounds. Numerous varieties of MOC systems have been proposed, mainly focusing on the construction of these multi-organ models, while there are only few studies on how to realize continual, automated, and stable testing, which still remains a significant challenge in the development process of MOCs. Herein, this review emphasizes the recent advancements in realizing long-term testing of MOCs to promote their capability for real-time monitoring of multi-organ interactions and chronic cellular reactions more accurately and steadily over the available chip models. Efforts in this field are still ongoing for better performance in the assessment of preclinical attributes for a new chemical entity. Further, we give a brief overview on the various biomedical applications of long-term testing in MOCs, including several proposed applications and their potential utilization in the future. Finally, we summarize with perspectives.

Experimental sepsis induces sustained inflammation and acetylcholinesterase activity impairment in the hypothalamus.

Sepsis is one of the leading causes of mortality in intensive care units besides causing profound alterations in the brain. One of the structures notably affected during sepsis is the hypothalamus, resulting in important physiopathological consequences. Recently, we provided evidence that the presence of neuroinflammation, oxidative stress, and apoptosis in the hypothalamus of septic rats, is accompanied by impairment of arginine vasopressin (AVP) secretion. We had also demonstrated that sepsis survivor animals present attenuated AVP secretion after osmotic challenge, suggesting a persistent inflammation in the hypothalamus. However, the long-term course of inflammation in the hypothalamus remains unclear. Thus, we induced sepsis by cecal ligation and puncture (CLP) in Wistar rats and, five days after sepsis induction, the hypothalamus of each animal was collected for analysis. Nonmanipulated animals (naive) were used as controls. We found that CLP-induced morphological alterations in microglial cells are accompanied by an increase in Iba-1 immunoreactivity. Moreover, we observed enhanced expression of NF-κB and CREB transcription factors, which are well known to modulate the immune response. Additionally, we found that phosphorylation of GSK3α/ß (a kinase upstream to the CREB signaling pathway) was increased, as well as COX-2, iNOS, and IL-6 that are canonic inflammatory proteins. Thus, our results indicated the presence of sustained activation of resident glial cells that may result in neuroinflammation and cholinergic neurotransmission disruptions in the hypothalamus.

Effect of erythropoietin on the migration of bone marrow-derived mesenchymal stem cells to the acute kidney injury microenvironment.

Bone marrow-derived mesenchymal stem cells (BMSCs) preferentially migrate to the injured tissue but with limited efficiency. Here we investigated the effect of erythropoietin (EPO) treatment on the BMSC migration to the acute kidney injury (AKI) microenvironment. The possible mechanisms were also discussed. A hypoxia/re-oxygenation (HR) model of renal tubular epithelial cells (RTECs) was established to generate AKI in vitro, and a chemotaxis experiment was conducted using the transwell chamber. EPO treatment enhanced the BMSC migration to the HR-RTEC culturing chamber in a SDF-1 level-dependent manner, which was fully inhibited by the treatment of anti-SDF-1 antibody. The BMSC migration could also be partly blocked by LY294002 (phosphoinositide 3-kinase (PI3K) inhibitor) and PD98059 (MAPK inhibitor). Western blot analysis showed that phosphorylated Akt and phosphorylated MAPK in BMSCs were enhanced by EPO treatment. In the in vivo experiment, BMSCs were transplanted into the AKI mice and EPO was subcutaneously injected. The results showed that EPO injection increased the SDF-1 protein expression and BMSC accumulation in the renal tissue, which was consistent with a decent improvement of renal function. In addition, the BMSC accumulation in the renal tissue was blocked by anti-SDF-1 antibody, LY294002 or PD98059. Our data suggest that AKI microenvironment had a directional chemotactic effect on BMSCs, which could be further enhanced by the EPO treatment. The increased SDF-1 level in the AKI microenvironment and the activations of PI3K/AKT and MAPK in BMSCs were the possible mechanisms for the effect of EPO. Therefore, BMSC transplantation combined with EPO injection can be a novel and effective approach for AKI repair.

Electromagnetic fields enhance chondrogenesis of human adipose-derived stem cells in a chondrogenic microenvironment in vitro.

We tested the hypothesis that electromagnetic field (EMF) stimulation enhances chondrogenesis in human adipose-derived stem cells (ADSCs) in a chondrogenic microenvironment. A two-dimensional hyaluronan (HA)-coated well (2D-HA) and a three-dimensional pellet culture system (3D-pellet) were used as chondrogenic microenvironments. The ADSCs were cultured in 2D-HA or 3D-pellet, and then treated with clinical-use pulse electromagnetic field (PEMF) or the innovative single-pulse electromagnetic field (SPEMF) stimulation. The cytotoxicity, cell viability, and chondrogenic and osteogenic differentiations were analyzed after PEMF or SPEMF treatment. The modules of PEMF and SPEMF stimulations used in this study did not cause cytotoxicity or alter cell viability in ADSCs. Both PEMF and SPEMF enhanced the chondrogenic gene expression (SOX-9, collagen type II, and aggrecan) of ADSCs cultured in 2D-HA and 3D-pellet. The expressions of bone matrix genes (osteocalcin and collagen type I) of ADSCs were not changed after SPEMF treatment in 2D-HA and 3D-pellet; however, they were enhanced by PEMF treatment. Both PEMF and SPEMF increased the cartilaginous matrix (sulfated glycosaminoglycan) deposition of ADSCs. However, PEMF treatment also increased mineralization of ADSCs, but SPEMF treatment did not. Both PEMF and SPEMF enhanced chondrogenic differentiation of ADSCs cultured in a chondrogenic microenvironment. SPEMF treatment enhanced ADSC chondrogenesis, but not osteogenesis, when the cells were cultured in a chondrogenic microenvironment. However, PEMF enhanced both osteogenesis and chondrogenesis under the same conditions. Thus the combination of a chondrogenic microenvironment with SPEMF stimulation can promote chondrogenic differentiation of ADSCs and may be applicable to articular cartilage tissue engineering.