Micropatterns of double-layered nanofiber scaffolds with dual functions of cell patterning and metabolite detection.

This paper describes the development of multi-functional nanofiber scaffolds consisting of multiple layers of nanofiber scaffolds and nanofiber-incorporated poly(ethylene glycol) (PEG) hydrogels. As a proof-of-concept demonstration, we fabricated micropatterned polymeric nanofiber scaffolds that were capable of simultaneously generating cellular micropatterns within a biomimetic environment and detecting cellular metabolic products within well-defined microdomains. To achieve this goal, we designed nanofiber scaffolds with both vertical and lateral microdomains. Vertically heterogeneous structures that were responsible for multi-functionality were realized by preparing double-layered nanofiber scaffolds consisting of an antibody-immobilized bottom layer of nanofibers and an upper layer of bare polystyrene (PS) nanofibers by a two-step sequential electrospinning process. Photopatterning of poly(ethylene glycol) (PEG) hydrogel on the electrospun nanofibers produced laterally heterogeneous micropatterned nanofiber scaffolds made of hydrogel microwells filled with a nanofibrous region, which is capable of generating cell and protein micropatterns due to the different interactions that cells and proteins have with PEG hydrogels and nanofibers. When HepG2 cells were seeded into resultant nanofiber scaffolds, cells selectively adhered within the 200 μm × 200 μm PS fiber microdomain and formed 180.2 ± 6.7 μm spheroids after 5 days of culture in the upper layer. Furthermore, immobilized anti-albumin in the bottom layer detected albumin secreted by micropatterned HepG2 cells with higher sensitivity than flat PS substrates, demonstrating successful accomplishment of dual functions using micropatterned double-layered nanofiber scaffolds.

[1]  S. Ramakrishna,et al.  In vitro study of human vascular endothelial cell function on materials with various surface roughness. , 2004, Journal of biomedical materials research. Part A.

[2]  Michael V. Pishko,et al.  Biomems Materials and Fabrication Technology: Control of Mammalian Cell and Bacteria Adhesion on Substrates Micropatterned with Poly(ethylene Glycol) Hydrogels , 2022 .

[3]  Milan Mrksich,et al.  Micropatterned Surfaces for Control of Cell Shape, Position, and Function , 1998, Biotechnology progress.

[4]  P. Carmeliet,et al.  Angiogenesis in cancer and other diseases , 2000, Nature.

[5]  Julian H. George,et al.  Exploring and Engineering the Cell Surface Interface , 2005, Science.

[6]  S. Agarwal,et al.  Use of electrospinning technique for biomedical applications , 2008 .

[7]  Katja Schenke-Layland,et al.  Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering. , 2008, Biomaterials.

[8]  T. McDevitt,et al.  Microsphere size effects on embryoid body incorporation and embryonic stem cell differentiation. , 2010, Journal of biomedical materials research. Part A.

[9]  Jungbae Kim,et al.  Preparation of biocatalytic nanofibres with high activity and stability via enzyme aggregate coating on polymer nanofibres , 2005, Nanotechnology.

[10]  Chad A Mirkin,et al.  The evolution of dip-pen nanolithography. , 2004, Angewandte Chemie.

[11]  Zhangqi Feng,et al.  Chitosan nanofiber scaffold enhances hepatocyte adhesion and function , 2009, Biotechnology Letters.

[12]  Hongwei Ma,et al.  Electrospinning of poly(dimethylsiloxane)/poly(methyl methacrylate) nanofibrous membrane: fabrication and application in protein microarrays. , 2009, Biomacromolecules.

[13]  M. Textor,et al.  Surface engineering approaches to micropattern surfaces for cell-based assays. , 2006, Biomaterials.

[14]  M. Toner,et al.  Microengineering of cellular interactions. , 2000, Annual review of biomedical engineering.

[15]  W. Park,et al.  Formation of silk fibroin matrices with different texture and its cellular response to normal human keratinocytes. , 2004, International journal of biological macromolecules.

[16]  Nicolas H Voelcker,et al.  Surface manipulation of biomolecules for cell microarray applications. , 2006, Trends in biotechnology.

[17]  Cato T Laurencin,et al.  Electrospun nanofibrous structure: a novel scaffold for tissue engineering. , 2002, Journal of biomedical materials research.

[18]  J Christopher Love,et al.  Multidimensional analysis of the frequencies and rates of cytokine secretion from single cells by quantitative microengraving. , 2010, Lab on a chip.

[19]  Won-Gun Koh,et al.  Fabrication of hydrogel-micropatterned nanofibers for highly sensitive microarray-based immunosensors having additional enzyme-based sensing capability , 2011 .

[20]  Y. Ito,et al.  Surface micropatterning to regulate cell functions. , 1999, Biomaterials.

[21]  K. Wilson,et al.  Characterisation of electrospun polystyrene scaffolds for three-dimensional in vitro biological studies. , 2006, Biomaterials.

[22]  Mingjun Zhang,et al.  Bio-Microarray Fabrication Techniques—A Review , 2006, Critical reviews in biotechnology.

[23]  Andreas Greiner,et al.  Electrospinning: a fascinating method for the preparation of ultrathin fibers. , 2007, Angewandte Chemie.

[24]  A. Khademhosseini,et al.  Controlling size, shape and homogeneity of embryoid bodies using poly(ethylene glycol) microwells. , 2007, Lab on a chip.

[25]  C. Schmidt,et al.  Simple benchtop patterning of hydrogel grids for living cell microarrays. , 2010, Lab on a chip.

[26]  Yonehiro Kanemura,et al.  Effect of microwell chip structure on cell microsphere production of various animal cells. , 2010, Journal of bioscience and bioengineering.

[27]  M. Gassmann,et al.  Central Necrosis in Isolated Hypoxic Human Pancreatic Islets: Evidence for Postisolation Ischemia , 2005, Cell transplantation.

[28]  G. Whitesides,et al.  Patterning proteins and cells using soft lithography. , 1999, Biomaterials.

[29]  Taiji Sohmura,et al.  Mass Fabrication of Small Cell Spheroids by Using Micro‐patterned Tissue Culture Plate , 2009 .

[30]  J. Southgate,et al.  Towards control of smooth muscle cell differentiation in synthetic 3D scaffolds. , 2008, Biomaterials.

[31]  Ning‐Ping Huang,et al.  The effect of nanofibrous galactosylated chitosan scaffolds on the formation of rat primary hepatocyte aggregates and the maintenance of liver function. , 2009, Biomaterials.

[32]  Keitaro Yoshimoto,et al.  Inverted pattern formation of cell microarrays on poly(ethylene glycol) (PEG) gel patterned surface and construction of hepatocyte spheroids on unmodified PEG gel microdomains. , 2009, Lab on a chip.

[33]  Michelle K. Leach,et al.  Rat hepatocyte aggregate formation on discrete aligned nanofibers of type-I collagen-coated poly(L-lactic acid). , 2010, Biomaterials.

[34]  K. Nakazawa,et al.  Two-dimensional microarray of HepG2 spheroids using collagen/polyethylene glycol micropatterned chip , 2008, Journal of materials science. Materials in medicine.

[35]  Younan Xia,et al.  Putting Electrospun Nanofibers to Work for Biomedical Research. , 2008, Macromolecular rapid communications.

[36]  A. Chilkoti,et al.  Micropatterning biological molecules on a polymer surface using elastomeric microwells. , 2001, Journal of the American Chemical Society.

[37]  Takashi Sugawara,et al.  Development of a novel protein fixation method with micron-order precision , 1995 .

[38]  Laura Marcu,et al.  Enzyme-containing hydrogel micropatterns serving a dual purpose of cell sequestration and metabolite detection. , 2009, Biosensors & bioelectronics.

[39]  Won-Gun Koh,et al.  Micropatterned Fibrous Scaffolds Fabricated Using Electrospinning and Hydrogel Lithography: New Platforms to Create Cellular Micropatterns , 2010 .

[40]  Junji Fukuda,et al.  Novel hepatocyte culture system developed using microfabrication and collagen/polyethylene glycol microcontact printing. , 2006, Biomaterials.

[41]  Tingrui Pan,et al.  Integrating sensing hydrogel microstructures into micropatterned hepatocellular cocultures. , 2009, Langmuir : the ACS journal of surfaces and colloids.

[42]  T. Park,et al.  Integration of Cell Culture and Microfabrication Technology , 2003, Biotechnology progress.

[43]  Seeram Ramakrishna,et al.  Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold. , 2005, Biomaterials.

[44]  Teruo Okano,et al.  Two‐Dimensional Multiarray Formation of Hepatocyte Spheroids on a Microfabricated PEG‐Brush Surface , 2004, Chembiochem : a European journal of chemical biology.

[45]  O. Kwon,et al.  Nanofabrication of microbial polyester by electrospinning promotes cell attachment , 2004 .

[46]  François Chatelain,et al.  Photochemical patterning of biological molecules inside a glass capillary. , 2005, Analytical chemistry.

[47]  R. Jackeray,et al.  Surface modification of polyacrylonitrile fiber for immobilization of antibodies and detection of analyte. , 2009, Analytica chimica acta.

[48]  A. Abbott.  Cell culture: Biology's new dimension , 2003, Nature.