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      Enhanced cryopreservation of MSCs in microfluidic bioreactor by regulated shear flow

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          Abstract

          Cell-matrix systems can be stored for longer period of time by means of cryopreservation. Cell-matrix and cell-cell interaction has been found to be critical in a number of basic biological processes. Tissue structure maintenance, cell secretary activity, cellular migration, and cell-cell communication all exist because of the presence of cell interactions. This complex and co-ordinated interaction between cellular constituents, extracellular matrix and adjacent cells has been identified as a significant contributor in the overall co-ordination of tissue. The prime objective of this investigation is to evaluate the effects of shear-stress and cell-substrate interaction in successful recovery of adherent human mesenchymal-stem-cells (hMSCs). A customized microfluidic bioreactor has been used for the purpose. We have measured the changes in focal-point-adhesion (FPAs) by changing induced shear stress inside the bioreactor. The findings indicate that with increase in shear stress, FPAs increases between substrate and MSCs. Further, experimental results show that increased FPAs (4e-3 μbar) enhances the cellular survivability of adherent MSCs. Probably, for the first time involvement of focal point interaction in the outcome of cryopreservation of MSCs has been clarified, and it proved a potentially new approach for modification of cryopreservation protocol by up-regulating focal point of cells to improve its clinical application.

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          Most cited references24

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          Focal Contacts as Mechanosensors

          The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.
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            Versatile, fully automated, microfluidic cell culture system.

            There is increasing demand for automated and quantitative cell culture technology, driven both by the intense activity in stem cell biology and by the emergence of systems biology. We built a fully automated cell culture screening system based on a microfluidic chip that creates arbitrary culture media formulations in 96 independent culture chambers and maintains cell viability for weeks. Individual culture conditions are customized in terms of cell seeding density, composition of culture medium, and feeding schedule, and each chamber is imaged with time-lapse microscopy. Using this device, we perform the first quantitative measurements of the influence of transient stimulation schedules on the proliferation, osteogenic differentiation, and motility of human primary mesenchymal stem cells.
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              A mechano-regulation model for tissue differentiation during fracture healing: analysis of gap size and loading.

              Bone has a capability to repair itself when it is fractured. Repair involves the generation of intermediate tissues, such as fibrous connective tissue, cartilage and woven bone, before final bone healing can occur. The intermediate tissues serve to stabilise the mechanical environment and provide a scaffold for differentiation of new tissues. The repair process is fundamentally affected by mechanical loading and by the geometric configuration of the fracture fragments. Biomechanical analyses of fracture healing have previously computed the stress distribution within the callus and identified the components of the stress tensor favouring or inhibiting differentiation of particular tissue phenotypes. In this paper, a biphasic poroelastic finite element model of a fracture callus is used to simulate the time-course of tissue differentiation during fracture healing. The simulation begins with granulation tissue (post-inflammation phase) and finishes with bone resorption. The biomechanical regulatory model assumes that tissue differentiation is controlled by a combination of shear strain and fluid flow acting within the tissue. High shear strain and fluid flows are assumed to deform the precursor cells stimulating formation of fibrous connective tissue, lower levels stimulate formation of cartilage, and lower again allows ossification. This mechano-regulatory scheme was tested by simulating healing in fractures with different gap sizes and loading magnitudes. The appearance and disappearance of the various tissues found in a callus was similar to histological observation. The effect of gap size and loading magnitude on the rate of reduction of the interfragmentary strain was sufficiently close to confirm the hypothesis that tissue differentiation phenomena could be governed by the proposed mechano-regulation model.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                17 October 2016
                2016
                : 6
                : 35416
                Affiliations
                [1 ]Department of Biomedical Engineering, National Institute of Technology , Raipur, India
                [2 ]Department of Biochemistry, University of Allahabad , Allahabad, India
                [3 ]Department of Biochemistry, Pt. JNM Medical College , Raipur, India
                Author notes
                Article
                srep35416
                10.1038/srep35416
                5066325
                27748463
                42ea49b1-42fb-4a16-8bfb-27d4913e3e0b
                Copyright © 2016, The Author(s)

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 22 June 2016
                : 02 September 2016
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