Quantitative Stain-Free and Continuous Multimodal Monitoring of Wound Healing In Vitro with Digital Holographic Microscopy

Impaired epithelial wound healing has significant pathophysiological implications in several conditions including gastrointestinal ulcers, anastomotic leakage and venous or diabetic skin ulcers. Promising drug candidates for accelerating wound closure are commonly evaluated in in vitro wound assays. However, staining procedures and discontinuous monitoring are major drawbacks hampering accurate assessment of wound assays. We therefore investigated digital holographic microscopy (DHM) to appropriately monitor wound healing in vitro and secondly, to provide multimodal quantitative information on morphological and functional cell alterations as well as on motility changes upon cytokine stimulation. Wound closure as reflected by proliferation and migration of Caco-2 cells in wound healing assays was studied and assessed in time-lapse series for 40 h in the presence of stimulating epidermal growth factor (EGF) and inhibiting mitomycin c. Therefore, digital holograms were recorded continuously every thirty minutes. Morphological changes including cell thickness, dry mass and tissue density were analyzed by data from quantitative digital holographic phase microscopy. Stimulation of Caco-2 cells with EGF or mitomycin c resulted in significant morphological changes during wound healing compared to control cells. In conclusion, DHM allows accurate, stain-free and continuous multimodal quantitative monitoring of wound healing in vitro and could be a promising new technique for assessment of wound healing.

[1]  J. Chiu,et al.  Modulation of chemotactic and pro-inflammatory activities of endothelial progenitor cells by hepatocellular carcinoma. , 2012, Cellular signalling.

[2]  C. Reutelingsperger,et al.  Annexin A1, formyl peptide receptor, and NOX1 orchestrate epithelial repair. , 2013, The Journal of clinical investigation.

[3]  Zhuo Wang,et al.  Optical measurement of cycle-dependent cell growth , 2011, Proceedings of the National Academy of Sciences.

[4]  J. Walsh,et al.  Peptic ulcer pathophysiology. , 1991, The Medical clinics of North America.

[5]  R. R. Bürk.  A factor from a transformed cell line that affects cell migration. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[6]  F. Drago,et al.  Effects of COOH–Terminal Tripeptide –MSH11–13 on Corneal Epithelial Wound Healing: Role of Nitric Oxide , 2006 .

[7]  D. Dirksen,et al.  Autofocusing in digital holographic phase contrast microscopy on pure phase objects for live cell imaging. , 2008, Applied optics.

[8]  Sugunavathi Sepramaniam,et al.  Water and Ion Channels: Crucial in the Initiation and Progression of Apoptosis in Central Nervous System? , 2008, Current neuropharmacology.

[9]  M. Tomikawa,et al.  Gastrointestinal mucosal regeneration: role of growth factors. , 1999, Frontiers in bioscience : a journal and virtual library.

[10]  Patrik Langehanenberg,et al.  Integral refractive index determination of living suspension cells by multifocus digital holographic phase contrast microscopy. , 2007, Journal of biomedical optics.

[11]  Joachim Wegener,et al.  Electrical wound-healing assay for cells in vitro. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Neurath,et al.  Assessment of tumor development and wound healing using endoscopic techniques in mice. , 2010, Gastroenterology.

[13]  B. Kemper,et al.  Survivin, a target to modulate the radiosensitivity of Ewing’s sarcoma , 2012, Strahlentherapie und Onkologie.

[14]  YoungJu Jo,et al.  Quantitative Phase Imaging Techniques for the Study of Cell Pathophysiology: From Principles to Applications , 2013, Sensors.

[15]  M. Basson,et al.  Differential Effects of Mucosal pH on Human (Caco-2) Intestinal Epithelial Cell Motility, Proliferation, and Differentiation , 1998, Digestive Diseases and Sciences.

[16]  Gabriel Popescu,et al.  Optical imaging of cell mass and growth dynamics. , 2008, American journal of physiology. Cell physiology.

[17]  D. Podolsky,et al.  Transforming growth factor β regulation of migration in wounded rat intestinal epithelial monolayers , 1993 .

[18]  Yongkeun Park,et al.  Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum , 2008, Proceedings of the National Academy of Sciences.

[19]  R. Coffey,et al.  Roles for transforming growth factor-alpha in the stomach. , 1995, Journal of clinical gastroenterology.

[20]  Christian Depeursinge,et al.  Early Cell Death Detection with Digital Holographic Microscopy , 2012, PloS one.

[21]  Patrik Langehanenberg,et al.  Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy. , 2010, Journal of biomedical optics.

[22]  F. Drago,et al.  Effects of the COOH-terminal tripeptide alpha-MSH(11-13) on corneal epithelial wound healing: role of nitric oxide. , 2006, Experimental eye research.

[23]  L. Thim,et al.  Trefoil peptides promote epithelial migration through a transforming growth factor beta-independent pathway. , 1994, The Journal of clinical investigation.

[24]  D. W. Ross.  The nature of unbalanced cell growth caused by cytotoxic agents , 1981, Virchows Archiv. B, Cell pathology including molecular pathology.

[25]  Patrik Langehanenberg,et al.  Atrial natriuretic peptide and nitric oxide signaling antagonizes vasopressin-mediated water permeability in inner medullary collecting duct cells. , 2009, American journal of physiology. Renal physiology.

[26]  H. Cross,et al.  Inhibition of sucrose-isomaltase expression by EGF in the human colon adenocarcinoma cells Caco-2. , 1991, The American journal of physiology.

[27]  C. Jumarie,et al.  Caco‐2 cells cultured in serum‐free medium as a model for the study of enterocytic differentiation in vitro , 1991, Journal of cellular physiology.

[28]  W. Bishop,et al.  Regulation of Caco-2 cell proliferation by basolateral membrane epidermal growth factor receptors. , 1994, The American journal of physiology.

[29]  Daniel Carl,et al.  Parameter-optimized digital holographic microscope for high-resolution living-cell analysis. , 2004, Applied optics.

[30]  S. Wehner,et al.  The effect of sealing with a fixed combination of collagen matrix-bound coagulation factors on the healing of colonic anastomoses in experimental high-risk mice models , 2010, Langenbeck's Archives of Surgery.

[31]  E. Hoffmann,et al.  Physiology of cell volume regulation in vertebrates. , 2009, Physiological reviews.

[32]  M. Basson,et al.  Extracellular pressure stimulates colon cancer cell proliferation via a mechanism requiring PKC and tyrosine kinase signals , 2004, Cell proliferation.

[33]  Melanie Boerries,et al.  Boolean approach to signalling pathway modelling in HGF-induced keratinocyte migration , 2012, Bioinform..

[34]  B. Kemper,et al.  Digital holographic microscopy quantifies the degree of inflammation in experimental colitis. , 2013, Integrative biology : quantitative biosciences from nano to macro.

[35]  N. Perkins,et al.  The role of RelA (p65) threonine 505 phosphorylation in the regulation of cell growth, survival, and migration , 2011, Molecular biology of the cell.

[36]  Chun-Min Lo,et al.  High-resolution quantitative phase-contrast microscopy by digital holography. , 2005, Optics express.

[37]  R. Barer.  Interference Microscopy and Mass Determination , 1952, Nature.

[38]  C. Jobin,et al.  PI3K-Dependent GSK3ß(Ser9)-Phosphorylation Is Implicated in the Intestinal Epithelial Cell Wound-Healing Response , 2011, PloS one.

[39]  P. J. Harris,et al.  Quantitative phase microscopy: a new tool for measurement of cell culture growth and confluency in situ , 2004, Pflügers Archiv.

[40]  Anne E Carpenter,et al.  CellProfiler: image analysis software for identifying and quantifying cell phenotypes , 2006, Genome Biology.

[41]  A. Blais,et al.  Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon , 2005, The Journal of Membrane Biology.

[42]  Christian Depeursinge,et al.  Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy. , 2009, Journal of biomedical optics.

[43]  J. Steinkamp,et al.  RAPID, SIMULTANEOUS MEASUREMENT OF DNA, PROTEIN, AND CELL VOLUME IN SINGLE CELLS FROM LARGE MAMMALIAN CELL POPULATIONS , 1973, The Journal of cell biology.

[44]  Stephanie Wu,et al.  Wound care: the role of advanced wound-healing technologies. , 2010, Journal of the American Podiatric Medical Association.

[45]  Ç. Şakalar,et al.  Pronounced transcriptional regulation of apoptotic and TNF–NF-kappa-B signaling genes during the course of thymoquinone mediated apoptosis in HeLa cells , 2013, Molecular and Cellular Biochemistry.

[46]  T. Dierks,et al.  The SULFs, Extracellular Sulfatases for Heparan Sulfate, Promote the Migration of Corneal Epithelial Cells during Wound Repair , 2013, PloS one.

[47]  Marjana Tomic-Canic,et al.  Cellular and molecular basis of wound healing in diabetes. , 2007, The Journal of clinical investigation.

[48]  Ping Wang,et al.  Inhibition of gap junction channel attenuates the migration of breast cancer cells , 2011, Molecular Biology Reports.

[49]  Christian Depeursinge,et al.  Determination of Transmembrane Water Fluxes in Neurons Elicited by Glutamate Ionotropic Receptors and by the Cotransporters KCC2 and NKCC1: A Digital Holographic Microscopy Study , 2011, The Journal of Neuroscience.

[50]  M. Basson,et al.  Repetitive deformation and pressure activate small bowel and colonic mucosal tyrosine kinase activity in vivo. , 2002, Metabolism: clinical and experimental.

[51]  M. Basson,et al.  Supraphysiologic extracellular pressure inhibits intestinal epithelial wound healing independently of luminal nutrient flow. , 2008, American journal of surgery.

[52]  Daniel Carl,et al.  Investigation of living pancreas tumor cells by digital holographic microscopy. , 2006, Journal of biomedical optics.

[53]  Jie Zhao,et al.  Morphological Measurement of Living Cells in Methanol with Digital Holographic Microscopy , 2013, Comput. Math. Methods Medicine.

[54]  Zhuo Wang,et al.  Tissue refractive index as marker of disease. , 2011, Journal of biomedical optics.

[55]  E. Cuche,et al.  Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy. , 2005, Optics letters.

[56]  Bertrand Simon,et al.  High‐resolution tomographic diffractive microscopy of biological samples , 2010, Journal of biophotonics.

[57]  A. Baldwin,et al.  Wound‐induced p38MAPK‐dependent histone H3 phosphorylation correlates with increased COX‐2 expression in enterocytes , 2006, Journal of cellular physiology.

[58]  Jérôme Parent,et al.  Label-free cytotoxicity screening assay by digital holographic microscopy. , 2013, Assay and drug development technologies.

[59]  Jochen Guck,et al.  Bacterial infection of macrophages induces decrease in refractive index , 2013, Journal of biophotonics.

[60]  C. Liang,et al.  In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro , 2007, Nature Protocols.

[61]  Anne E Carpenter,et al.  CellProfiler: free, versatile software for automated biological image analysis. , 2007, BioTechniques.

[62]  Patrik Langehanenberg,et al.  Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial cells , 2010, Thrombosis and Haemostasis.

[63]  A. Lloyd,et al.  The Regulation of Cell Size , 2013, Cell.

[64]  Steffi Ketelhut,et al.  Sensing dynamic cytoplasm refractive index changes of adherent cells with quantitative phase microscopy using incorporated microspheres as optical probes , 2012, Journal of biomedical optics.