Two-dimensional microarray of HepG2 spheroids using collagen/polyethylene glycol micropatterned chip

A new cell chip technology in the form of a two-dimensional microarray of HepG2 spheroids was developed by using the microcontact printing technique. The chip consisted of several collagen spots in a triangular arrangement within a 100-mm2 area at the center of a glass plate (24 × 24 mm), which served as the cell adhesion area; the region excluding the collagen spots that was modified with polyethylene glycol (PEG) served as the non-adhesion area. HepG2 cells inoculated onto the chip gradually formed spheroids with smooth surfaces and high circularity on each collagen spot due to cell proliferation; the spheroid diameters remained constant after 10 days of culture. Such a two-dimensional microarray configuration of HepG2 spheroids could be maintained for at least 2 weeks. The spheroid diameter was directly proportional to the pitch between the collagen spots on the chip. This indicates that we can factitiously control the spheroid diameter. In addition, albumin secretion activity of HepG2 spheroids increased with the increase of spheroid diameter. This chip technology may be applicable as a cellular platform for developing two-dimensional spheroid microarrays.

[1]  Junji Fukuda,et al.  Orderly arrangement of hepatocyte spheroids on a microfabricated chip. , 2005, Tissue engineering.

[2]  Jinsheng Xu,et al.  Characterisation of some cytotoxic endpoints using rat liver and HepG2 spheroids as in vitro models and their application in hepatotoxicity studies. I. Glucose metabolism and enzyme release as cytotoxic markers. , 2003, Toxicology and applied pharmacology.

[3]  K. Asano,et al.  Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments. , 1990, Experimental cell research.

[4]  S. Bhatia,et al.  An extracellular matrix microarray for probing cellular differentiation , 2005, Nature Methods.

[5]  K. Sugimachi,et al.  Polyurethane Foam/Spheroid Culture System Using Human Hepatoblastoma Cell Line (Hep G2) as a Possible New Hybrid Artificial Liver , 2001, Cell transplantation.

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

[7]  Mehmet Toner,et al.  Designing a hepatocellular microenvironment with protein microarraying and poly(ethylene glycol) photolithography. , 2004, Langmuir : the ACS journal of surfaces and colloids.

[8]  G M Whitesides,et al.  Biological surface engineering: a simple system for cell pattern formation. , 1999, Biomaterials.

[9]  Kohji Nakazawa,et al.  Hepatocyte Spheroids in Polyurethane Foams: Functional Analysis and Application for a Hybrid Artificial Liver , 1998 .

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

[11]  H. Thierens,et al.  Screening for supra-additive effects of cytotoxic drugs and gamma irradiation in an in vitro model for hepatocellular carcinoma. , 2004, Canadian journal of physiology and pharmacology.

[12]  F. Alvarez,et al.  Long-term culture of adult rat hepatocyte spheroids. , 1992, Experimental cell research.

[13]  Yoshihiro Ito,et al.  Preparation of a protein micro-array using a photo-reactive polymer for a cell-adhesion assay. , 2003, Biomaterials.

[14]  H. Iwata,et al.  Parallel analysis of multiple surface markers expressed on rat neural stem cells using antibody microarrays. , 2005, Biomaterials.

[15]  L. Griffith,et al.  Functional behavior of primary rat liver cells in a three-dimensional perfused microarray bioreactor. , 2002, Tissue engineering.

[16]  Tomoyuki Yasukawa,et al.  A multicellular spheroid array to realize spheroid formation, culture, and viability assay on a chip. , 2007, Biomaterials.

[17]  Helene Andersson,et al.  Microfabrication and microfluidics for tissue engineering: state of the art and future opportunities. , 2004, Lab on a chip.

[18]  Junji Fukuda,et al.  Hepatocyte spheroid culture on a polydimethylsiloxane chip having microcavities , 2006, Journal of biomaterials science. Polymer edition.

[19]  Jean Philippe Stephan,et al.  Development of a frozen cell array as a high-throughput approach for cell-based analysis. , 2002, The American journal of pathology.

[20]  Ali Khademhosseini,et al.  Layer-by-layer deposition of hyaluronic acid and poly-L-lysine for patterned cell co-cultures. , 2004, Biomaterials.

[21]  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.

[22]  D. Sabatini,et al.  Microarrays of cells expressing defined cDNAs , 2001, Nature.

[23]  Ju Hun Yeon,et al.  Cytotoxicity test based on electrochemical impedance measurement of HepG2 cultured in microfabricated cell chip. , 2005, Analytical biochemistry.

[24]  C Selden,et al.  Human hepatocyte cell lines proliferating as cohesive spheroid colonies in alginate markedly upregulate both synthetic and detoxificatory liver function. , 2001, Journal of hepatology.

[25]  Martin Fussenegger,et al.  Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. , 2003, Biotechnology and bioengineering.

[26]  K Sugimachi,et al.  Efficacy of a Polyurethane Foam/Spheroid Artificial Liver by Using Human Hepatoblastoma Cell Line (Hep G2) , 2003, Cell transplantation.