Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions

Abstract

Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling cellular microenvironments including cell–matrix interactions, soluble stimuli and cell–cell interactions. Here, we present a novel approach to generate layered patterning of hepatocyte spheroids on micropatterned non-parenchymal feeder cells using microfabricated poly(ethylene glycol) (PEG) hydrogels. Micropatterned PEG-hydrogel-treated substrates with two-dimensional arrays of gelatin circular domains (ϕ = 100 μm) were prepared by photolithographic method. Only on the critical structure of PEG hydrogel with perfect protein rejection, hepatocytes were co-cultured with non-parenchymal cells to be led to enhanced hepatocyte functions. Then, we investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell–cell interactions. In particular, to elucidate the influence of soluble factors on hepatocyte function, hepatocyte spheroids underlaid with fibroblasts (NIH/3T3 mouse fibroblasts) or endothelial cells (BAECs: bovine aortic endothelial cells) were compared with physically separated co-culture of hepatocyte monospheroids with NIH3T3 or BAEC using trans-well culture systems. Our results suggested that direct heterotypic cell-to-cell contact and soluble factors, both of these between hepatocytes and fibroblasts, significantly enhanced hepatocyte functions. In contrast, direct heterotypic cell-to-cell contact between hepatocytes and endothelial cells only contributed to enhance hepatocyte functions. This patterning technique can be a useful experimental tool for applications in basic science, drug screening and tissue engineering, as well as in the design of artificial liver devices.

Engineering Multi‐Cellular Spheroids for Tissue Engineering and Regenerative Medicine

“…For example, cell adhesion molecules such as gelatin or collagen were used to coat a circular pattern onto a non-adherent surface to control the spheroid size, which was released with lysis by collagenase after cell assembly formation. [93,94] Hepatocytes and fibroblasts were formed as co-cultured spheroids with a uniform size of 100 µm on gelatin-patterned PEG, and the size of hepatocyte spheroids was controlled from 50 to 100 µm according to the size of circular patterns. [93,94] A polydopamine pattern was also applied for the high-throughput production of spheroids on temperature-responsive hydrogel by micro-contact printing.…”

“…[93,94] Hepatocytes and fibroblasts were formed as co-cultured spheroids with a uniform size of 100 µm on gelatin-patterned PEG, and the size of hepatocyte spheroids was controlled from 50 to 100 µm according to the size of circular patterns. [93,94] A polydopamine pattern was also applied for the high-throughput production of spheroids on temperature-responsive hydrogel by micro-contact printing. Using this platform, human MSCs and fibroblasts spheroids demonstrated uniform size (90-100 µm), with this uniformity maintained for 7 days.…”

“…Controlled size, simplicity [ 71,74] Automated hanging drop Labor-effectiveness, controlled size [75] Superhydrophobic surface Efficiency, controlled size [ 76,77] Insert formed by 3D printing Efficiency, controlled size [ 101] Centrifugal force Pellet culture Controlled size, simplicity [ 14,78] Spinner flask Mass production, reduced apoptosis [ 82,83] Rotary culture Mass production, controlled size [81] Surface chemistry Chemical patterning Mass production (<100 µm), [ 71,93,94,95,96,97] (PNIPAAm/biomolecules) controlled size Microstructure Topological pattern Simplicity, controlled size [ 40,89,90] Micro-well plate Controlled size [ 41,85,86,87,88] Flow-based automation Microfluidics Labor-effectiveness, controlled size, effective medium change [ 98,99,100] Acoustic wave Surface acoustic wave Controlled size [ 103,105] or centrifugal forces) for hanging drop and pellet culture methods, and biomaterials with microstructure and defined surface chemistry for the Aggrewell and the ultra-low attachment plate method, respectively. [14,16,71] These methods have their advantages and disadvantages, and thus, the method is dependent on the intended application.…”

Mimicking liver sinusoidal structures and functions using a 3D-configured microfluidic chip

“…In the past decades, in vitro two-dimensional (2D) or 3D liver models have been developed for understanding the pathophysiology, diagnosis and treatment of liver diseases, mainly applying the patterned HC culture alone or a co-culture of HCs with nonparenchymal cells (NPCs). These 2D or 3D models are likely to favor LSEC morphology and phenotype, [4][5][6] enhance albumin (ALB) and urea secretion, [4][5][6][7][8][9][10][11][12][13][14][15][16][17] promote cytochrome activity, [4][5][6][7][10][11][12] or augment bacteria susceptibility. 11 Fluid flow is another key factor to better mimic physiological environments in the sinusoid.…”

Whole liver engineering: A promising approach to develop functional liver surrogates

“…To reintroduce both parenchymal and non‐parenchymal cells in the decellularized liver scaffolds may be important for appropriate cell interactions. Establishing interactions between hepatocytes and non‐parenchymal cells have been shown to be beneficial to hepatocyte survival and functions . Co‐seeding hepatocytes with non‐parenchymal cells may be a favourable strategy to improve the hepatocyte functions in the decellularized liver scaffolds by re‐establishing proper cell‐cell interactions.…”