CARS and SHG microscopy to follow collagen production in living human corneal fibroblasts and mesenchymal stem cells in fibrin hydrogel 3D cultures

Coherent anti-Stokes Raman scattering (CARS) microscopy is used in conjunction with second harmonic generation (SHG) to follow the early stage of stem cell differentiation within a three-dimensional (3D) scaffold. Formation of an extracellular matrix mainly composed of collagen is one of the first signs of human mesenchymal stem cell (hMSC) differentiation. This work shows that the multimodal CARS and SHG constitutes a powerful noninvasive, label-free technique for monitoring collagen production in 3D cell cultures. Its simultaneous imaging of cell morphology and the distribution of the collagen produced by living cells during long-term (4 weeks) experiment furnished important information about the cell-scaffold interaction and the establishment of the extracellular matrix, while its very low collagen detection limit permitted mapping of even the small quantity produced in a few hours. This finding indicates that the multimodal CARS and SHG imaging can be proposed as a new way of monitoring the differentiation of stem cells by evaluating their production of collagen in both short and long-term experiments. Further evidence is also provided of the efficacy of fibrin hydrogel as a scaffold autoinducing the differentiation stimulus in 3D human mesenchymal stem cell cultures. Copyright © 2012 John Wiley & Sons, Ltd.

[1]  Annika Enejder,et al.  Visualization of the cellulose biosynthesis and cell integration into cellulose scaffolds. , 2010, Biomacromolecules.

[2]  R. Poulsom,et al.  Adult stem cell plasticity: new pathways of tissue regeneration become visible. , 2002, Clinical science.

[3]  Haiyang Yu,et al.  Osteogenic differentiation of bone marrow-derived mesenchymal stromal cells on bone-derived scaffolds: effect of microvibration and role of ERK1/2 activation. , 2011, European cells & materials.

[4]  Watt W Webb,et al.  Interpreting second-harmonic generation images of collagen I fibrils. , 2005, Biophysical journal.

[5]  W. R. Wiley,et al.  Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering , 1999 .

[6]  Antonios G Mikos,et al.  Biomimetic materials for tissue engineering. , 2003, Biomaterials.

[7]  Jochen Ringe,et al.  Stem cells for regenerative medicine: advances in the engineering of tissues and organs , 2002, Naturwissenschaften.

[8]  H. Cheung,et al.  Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds. , 2009, Stem cells and development.

[9]  Benjamin M. Wu,et al.  Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro. , 2006, Tissue engineering.

[10]  W. Webb,et al.  Nonlinear magic: multiphoton microscopy in the biosciences , 2003, Nature Biotechnology.

[11]  October I Physical Review Letters , 2022 .

[12]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[13]  F. Ganikhanov,et al.  Multimodal nonlinear optical imaging of collagen arrays. , 2008, Journal of structural biology.

[14]  William A Mohler,et al.  Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres. , 2006, Biophysical journal.

[15]  A. Caplan Adult mesenchymal stem cells for tissue engineering versus regenerative medicine , 2007, Journal of cellular physiology.

[16]  William A Mohler,et al.  Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. , 2002, Biophysical journal.

[17]  Moshe Levi,et al.  Multimodal CARS microscopy determination of the impact of diet on macrophage infiltration and lipid accumulation on plaque formation in ApoE-deficient mice[S] , 2010, Journal of Lipid Research.

[18]  T. Feser,et al.  Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells Cultured onto Three Different Polymers In Vitro , 2005, Annals of Biomedical Engineering.

[19]  B. Tromberg,et al.  Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Andrew Ridsdale,et al.  Multimodal nonlinear optical imaging of atherosclerotic plaque development in myocardial infarction-prone rabbits. , 2010, Journal of biomedical optics.

[21]  Arnold I Caplan,et al.  Isolation of human marrow-derived mesenchymal stem cells. , 2006, Experimental hematology.

[22]  Yoshinori Kuboki,et al.  Differentiation of mesenchymal stem cells into osteoblasts on honeycomb collagen scaffolds , 2006, Biotechnology and bioengineering.

[23]  D. English,et al.  Stem cells and development. , 2004, Stem cells and development.

[24]  J. Welter,et al.  Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells: tips and tricks. , 2011, Methods in molecular biology.

[25]  M. Pittenger,et al.  Multilineage potential of adult human mesenchymal stem cells. , 1999, Science.

[26]  Guy Cox,et al.  3-dimensional imaging of collagen using second harmonic generation. , 2003, Journal of structural biology.

[27]  P. Janmey,et al.  Tissue Cells Feel and Respond to the Stiffness of Their Substrate , 2005, Science.

[28]  X. Xie,et al.  Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy Published, JLR Papers in Press, August 16, 2003. DOI 10.1194/jlr.D300022-JLR200 , 2003, Journal of Lipid Research.

[29]  F. Canovas,et al.  Microenvironmental changes during differentiation of mesenchymal stem cells towards chondrocytes , 2007, Arthritis research & therapy.

[30]  S. Lowen The Biophysical Journal , 1960, Nature.

[31]  Shuangmu Zhuo,et al.  Visualization of collagen regeneration in mouse dorsal skin using second harmonic generation microscopy , 2009 .

[32]  E. Hall,et al.  The nature of biotechnology. , 1988, Journal of biomedical engineering.

[33]  Melinda Larsen,et al.  Extracellular matrix dynamics in development and regenerative medicine , 2008, Journal of Cell Science.

[34]  Adrian F. Pegoraro,et al.  Multimodal CARS microscopy of structured carbohydrate biopolymers , 2010, Biomedical optics express.

[35]  A. Hall Applied Optics. , 2022, Science.

[36]  Giselle Chamberlain,et al.  Concise Review: Mesenchymal Stem Cells: Their Phenotype, Differentiation Capacity, Immunological Features, and Potential for Homing , 2007, Stem cells.

[37]  Hannu Kautiainen,et al.  Arthritis Research & Therapy , 2009 .

[38]  Anthony M. D. Lee,et al.  Spectroscopic characterization and microscopic imaging of extracted and in situ cutaneous collagen and elastic tissue components under two‐photon excitation , 2009, Skin research and technology : official journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging.

[39]  R. Franceschi,et al.  Relationship between collagen synthesis and expression of the osteoblast phenotype in MC3T3‐E1 cells , 1992, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[40]  P. Janmey,et al.  Fibrin gels and their clinical and bioengineering applications , 2009, Journal of The Royal Society Interface.

[41]  D. Prockop,et al.  Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. , 2006, Cytotherapy.

[42]  T. Nishida,et al.  Interactions of extracellular collagen and corneal fibroblasts: Morphologic and biochemical changes of rabbit corneal cells cultured in a collagen matrix , 1988, In Vitro Cellular & Developmental Biology.

[43]  S. Sen,et al.  Matrix Elasticity Directs Stem Cell Lineage Specification , 2006, Cell.

[44]  Benjamin Wu,et al.  Modulation of 3D fibrin matrix stiffness by intrinsic fibrinogen-thrombin compositions and by extrinsic cellular activity. , 2009, Tissue engineering. Part A.

[45]  S. Böhm,et al.  Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC , 2011, Cell Communication and Signaling.

[46]  A. Beck‐Sickinger,et al.  Cell Communication and Signaling , 2009 .

[47]  M. Sefton,et al.  Tissue engineering. , 1998, Journal of cutaneous medicine and surgery.

[48]  Patrick Stoller,et al.  Quantitative second-harmonic generation microscopy in collagen. , 2003, Applied optics.

[49]  Theo H Smit,et al.  Scaffold Stiffness Influences Cell Behavior: Opportunities for Skeletal Tissue Engineering , 2008, The Open Orthopaedics Journal.

[50]  Watt,et al.  Journal of Cell Science , 1996, Nature.

[51]  L. Allen Stem cells. , 2003, The New England journal of medicine.

[52]  Zhao-Jun Liu,et al.  Trafficking and differentiation of mesenchymal stem cells , 2009, Journal of cellular biochemistry.

[53]  Gengfeng Zheng,et al.  Laser-scanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology. , 2002, Biophysical journal.

[54]  R. Misra,et al.  Biomaterials , 2008 .

[55]  L M Loew,et al.  Second-harmonic imaging microscopy of living cells. , 2001, Journal of biomedical optics.

[56]  Chen-Yuan Dong,et al.  The discrimination of type I and type II collagen and the label-free imaging of engineered cartilage tissue. , 2010, Biomaterials.