Biodegradable meshes printed with extracellular matrix proteins support micropatterned hepatocyte cultures

Abstract

The spatial organization of cells of different phenotypes is an important and often defining determinant of tissue function. In tissue engineering, which attempts to rebuild functional tissues from cellular and synthetic components, spatial patterning of cells onto biomaterials is likely to be equally important. We have printed combinatorial arrays of extracellular matrix (ECM) and screened them for attachment by HepG2 hepatocytes, LX-2 hepatic stellate cells, primary portal fibroblasts, and bovine aortic endothelial cells-cells selected as representative phenotypes found in adult liver. Differential cell attachment to the underlying matrix proteins allowed us to establish two-dimensional co-cultures of HepG2 with these non-parenchymal cell types. These general approaches were then translated to tissue engineering scaffolds where deposition of ECM proteins onto electrospun polylactide meshes resulted in patterned HepG2 cultures. We observed that the spatial organization of fibronectin deposits influenced HepG2 attachment and the establishment of co-cultures on our arrays. These micropatterned co-culture systems should serve as valuable tools for studying the soluble and insoluble signals involved in liver development, function, and disease.

FIG. 1.

Extracellular matrix protein microarray. (A) Diagram showing composition and position of all 64 unique compositions of collagen I, III, and IV; fibronectin (Fn); laminin (Ln); and Engelbreth-Holm-Swarm matrix (E). Number adjacent to each spot corresponds to the identity (ID) of the mixture in Table 1. Position of the pure Fn spot is indicated, and the box shows its nearest extracellular matrix (ECM) neighbors. (B) Diagram showing pattern of protein deposition for each of the six ECM proteins used to print the microarray. (C) Proteins were deposited in four replicates onto HydroGel microarray slides using a Virtek ChipWriter Pro robotic microarrayer.

FIG. 2.

Characterization of extracellular matrix (ECM) microarray. (A) Specified position and immunofluorescent image of an ECM microarray probed with a primary antibody against collagen I and using a secondary antibody conjugated to Alexafluor 633. Immunofluorescent image was obtained using a GenePix 4200A scanner. (B) Correlation of fluorescence intensity and rat collagen I concentration after protein printing (□) and following incubation (▪) for 24 h at 37°C in cell culture medium. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Viability of cultured cells on extracellular matrix (ECM) protein arrays. (A) Live/dead staining reveals viability of HepG2 cells on ECM combinatorial array. Co-localization of green (live) and red (dead) fluorescence results in a yellow signal. (B) Brightfield image (20×) of a single HepG2 island after exposure to Trypan blue. Brightfield images were obtained using an Olympus digital camera mounted on a Nikon TS100 microscope. Fluorescent images were obtained using a GenePix 4200A scanner. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Cell attachment to extracellular matrix (ECM) protein microarrays. (A) Representative images of HepG2 hepatocytes, LX-2 hepatic stellate cells (HSCs), portal fibroblasts, and bovine aortic endothelial cells cultured on the combinatorial ECM protein arrays. Cell cultures were established overnight, washed, fixed, and then permeabilized and stained using CyQuantNF. Cells were imaged on a GenePix 4200A fluorescent laser scanner. Yellow circle indicates position of fibronectin island. (B) The amount of cells occupying each ECM island was estimated by measuring the total cellular fluorescence and plotted as a function of the underlying ECM material. Cultured cells do not show patterns of differential attachment but HepG2 cells are observed to attach less to fibronectin spots than LX-2 HSCs or portal fibroblasts. Color images available online at www.liebertonline.com/ten.

FIG. 5.

Extracellular matrix (ECM) arrays support co-cultures of HepG2 cells with bovine aortic endothelial cells (BAECs) or LX-2 hepatic stellate cells (HSCs). (A) ECM microarrays support co-cultures of HepG2 hepatocytes were prestained with CellTracker Orange CMTMR (HepG2-CMTMRs) with BAECs or LX-2 HSCs. Sequential seeding with BAECs (middle panel) or LX-2 HSCs (right panel) onto the 64 ECM combinatorial protein arrays produced co-cultures with HepG2 cells, which do not attach to fibronectin (Fn) spots (left panel). (B) ECM arrays composed of alternating rows of Fn and rat collagen I were sequentially seeded with BAECs and HepG2-CMTMRs to establish co-cultures patterns. Brightfield images show the pattern and boundary of the cell cultures in the 4 × 4 array. Fluorescent images show that rows of rat collagen I (black arrows) are occupied by HepG2-CMTMRs and that rows of Fn are cultured with both cell types. (C) Magnified images of boxed regions from (B). Brightfield (black box) and fluorescence (yellow box) image showing a Fn island occupied mainly by BAECs. Brightfield images were obtained using an Olympus digital camera mounted on a Nikon TS100 microscope. Fluorescent images were acquired using an Olympus IX71 inverted fluorescent microscope. Color images available online at www.liebertonline.com/ten.

FIG. 6.

HepG2 hepatocytes cultured on electrospun biodegradable scaffolds. (A) Scanning electron microscopy (SEM) images of polylactic acid (PLA) fibers. (B) Average fiber diameter was determined from image analysis of SEM micrographs. (C) Brightfield (4×) and fluorescent image of PLA scaffold cultured with HepG2 hepatocytes pre-stained using CellTracker Orange CMTMR. Fluorescent image shows islands of HepG2 cells cultured into an 8 × 8 grid pattern. (D) SEM micrograph of an electrospun mesh of poly-lactic acid printed with extracellular matrix (ECM) and seeded with cells. Inset shows depression made on mesh after ECM printing using a manual arrayer. Scale bar of inset is 500 μm. Color images available online at www.liebertonline.com/ten.