Three‐dimensional nanocharacterization of porous hydrogel with ion and electron beams

Porous hydrogels provide an excellent environment for cell growth and tissue regeneration, with high permeability for oxygen, nutrients, and other water‐soluble metabolites through their high water‐content matrix. The ability to image three‐dimensional (3D) cell growth is crucial for understanding and studying various cellular activities in 3D context, particularly for designing new tissue engineering scaffold, but it is still challenging to study cell‐biomaterial interfaces with high resolution imaging. We demonstrate using focused ion beam (FIB) milling, electron imaging, and associated microanalysis techniques that novel 3D characterizations can be performed effectively on cells growing inside 3D hydrogel scaffold. With FIB‐tomography, the porous microstructures were revealed at nanometer resolution, and the cells grown inside. The results provide a unique 3D measurement of hydrogel porosity, as compared with those from porosimetry, and offer crucial insights into material factors affecting cell proliferation at specific regions within the scaffold. We also proved that high throughput correlative imaging of cell growth is viable through a silicon membrane based environment. The proposed approaches, together with the protocols developed, provide a unique platform for analysis of the microstructures of novel biomaterials, and for exploration of their interactions with the cells as well. Biotechnol. Bioeng. 2013; 110: 318–326. © 2012 Wiley Periodicals, Inc.

[1]  M. Milani,et al.  Comparison of different preparation methods of biological samples for FIB milling and SEM investigation , 2009, Journal of microscopy.

[2]  Andrew M. Minor,et al.  Focused Ion Beam Microscopy and Micromachining , 2007 .

[3]  Lucille A. Giannuzzi,et al.  Introduction to Focused Ion Beams , 2005 .

[4]  A. Cismak,et al.  Investigation of cell-substrate interactions by focused ion beam preparation and scanning electron microscopy. , 2011, Acta biomaterialia.

[5]  M. Kurisawa,et al.  Injectable biodegradable hydrogels with tunable mechanical properties for the stimulation of neurogenesic differentiation of human mesenchymal stem cells in 3D culture. , 2010, Biomaterials.

[6]  J. Guck,et al.  The cavity-to-cavity migration of leukaemic cells through 3D honey-combed hydrogels with adjustable internal dimension and stiffness. , 2010, Biomaterials.

[7]  Daniel Phifer,et al.  Two-dimensional and 3-dimensional analysis of bone/dental implant interfaces with the use of focused ion beam and electron microscopy. , 2007, Journal of oral and maxillofacial surgery : official journal of the American Association of Oral and Maxillofacial Surgeons.

[8]  B. Humbel,et al.  The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy. , 2010, Journal of structural biology.

[9]  M Epple,et al.  Uptake and intracellular distribution of silver nanoparticles in human mesenchymal stem cells. , 2011, Acta biomaterialia.

[10]  S. Hollister.  Porous scaffold design for tissue engineering , 2005, Nature materials.

[11]  S. Subramaniam,et al.  3D imaging of mammalian cells with ion-abrasion scanning electron microscopy. , 2009, Journal of structural biology.

[12]  M. Fay,et al.  An appraisal of ultramicrotomy, FIBSEM and cryogenic FIBSEM techniques for the sectioning of biological cells on titanium substrates for TEM investigation , 2009, Journal of microscopy.

[13]  M. Hayles,et al.  A technique for improved focused ion beam milling of cryo‐prepared life science specimens , 2007, Journal of microscopy.

[14]  Sriram Subramaniam,et al.  Correlative 3D imaging of whole mammalian cells with light and electron microscopy. , 2011, Journal of structural biology.

[15]  C A van Blitterswijk,et al.  Enzymatically-crosslinked injectable hydrogels based on biomimetic dextran-hyaluronic acid conjugates for cartilage tissue engineering. , 2010, Biomaterials.

[16]  W. Baumann,et al.  FIB preparation and SEM investigations for three-dimensional analysis of cell cultures on microneedle arrays. , 2012, Scanning.

[17]  Sputtering rate of micromilling on water ice with focused ion beam in a cryogenic environment , 2008 .

[18]  J. Mao,et al.  In vitro evaluation of macroporous hydrogels to facilitate stem cell infiltration, growth, and mineralization. , 2009, Tissue engineering. Part A.

[19]  M. Kurisawa,et al.  An injectable enzymatically crosslinked hyaluronic acid- hydrogel system with independent tuning of mechanical strength and gelation rate. , 2008, Soft matter.

[20]  G. Knott,et al.  Serial Section Scanning Electron Microscopy of Adult Brain Tissue Using Focused Ion Beam Milling , 2008, The Journal of Neuroscience.

[21]  Lucille A. Giannuzzi,et al.  Introduction to focused ion beams : instrumentation, theory, techniques, and practice , 2010 .

[22]  C B Sledge,et al.  Matrix collagen type and pore size influence behaviour of seeded canine chondrocytes. , 1997, Biomaterials.

[23]  R. Schalek,et al.  Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy , 2007, Nature Methods.

[24]  C. Burkhardt,et al.  Imaging of Cell‐to‐Material Interfaces by SEM after in situ Focused Ion Beam Milling on Flat Surfaces and Complex 3D‐Fibrous Structures , 2009 .

[25]  Donald Bliss,et al.  Ion-Abrasion Scanning Electron Microscopy Reveals Surface-Connected Tubular Conduits in HIV-Infected Macrophages , 2009, PLoS pathogens.

[26]  J. McGeoch.  Topology of the mammalian cell via cryo‐FIB etching , 2007, Journal of microscopy.

[27]  Qi Li,et al.  A macroporous hydrogel for the coculture of neural progenitor and endothelial cells to form functional vascular networks in vivo. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[28]  S. Joshi,et al.  A study of angular effects in focused ion beam milling of water ice , 2008 .

[29]  J. Planell,et al.  Focused ion beam/scanning electron microscopy characterization of cell behavior on polymer micro-/nanopatterned substrates: a study of cell-substrate interactions. , 2008, Micron.

[30]  F. Pérez-Willard,et al.  Focused ion beam (FIB) combined with high resolution scanning electron microscopy: a promising tool for 3D analysis of chromosome architecture. , 2009, Journal of structural biology.

[31]  Felix J. B. Bäuerlein,et al.  Micromachining tools and correlative approaches for cellular cryo-electron tomography. , 2010, Journal of structural biology.

[32]  Juhyoun Kwak,et al.  Ion-beam sculpting at nanometre length scales , 2001 .

[33]  B. Lee,et al.  In-situ injectable physically and chemically gelling NIPAAm-based copolymer system for embolization. , 2006, Biomacromolecules.

[34]  Bradley C. Lowekamp,et al.  3D visualization of HIV transfer at the virological synapse between dendritic cells and T cells , 2010, Proceedings of the National Academy of Sciences.

[35]  S. Subramaniam,et al.  Site-specific 3D imaging of cells and tissues with a dual beam microscope. , 2006, Journal of structural biology.

[36]  Sriram Subramaniam,et al.  3d Imaging of Diatoms with Ion-abrasion Scanning Electron Microscopy , 2022 .