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ABSTRACT: We have developed a three-dimensional (3D) liver-on-a-chip to investigate the interaction of hepatocytes and hepatic stellate cells (HSCs) in which primary 3D hepatocyte spheroids and HSCs are co-cultured without direct cell-cell contact. Here, we show that the 3D liver chip offers substantial advantages for the formation and harvesting of spheroids. The most important feature of this liver chip is that it enables continuous flow of medium to the cells through osmotic pumping, and thus requires only minimal handling and no external power source. We also demonstrate that flow assists the formation and long-term maintenance of spheroids. Additionally, we quantitatively and qualitatively investigated the paracrine effects of HSCs, demonstrating that HSCs assist in the maintenance of hepatocyte spheroids and play an important role in the formation of tight cell-cell contacts, thereby improving liver-specific function. Spheroids derived from co-cultures exhibited improved albumin and urea secretion rates compared to mono-cultured spheroids after 9 days. Immunostaining for cytochrome P450 revealed that the enzymatic activity of spheroids co-cultured for 8 days was greater than that of mono-cultured spheroids. These results indicate that this system has the potential for further development as a unique model for studying cellular interactions or as a tool that can be incorporated into other models aimed at creating hepatic structure and prolonging hepatocyte function in culture.
Lab on a Chip 05/2013; · 5.67 Impact Factor
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ABSTRACT: We have generated human hepatocyte spheroids with uniform size and shape by co-culturing 1∶1 mixtures of primary human hepatocytes (hHeps) from partial hepatectomy specimens and human adipose-derived stem cells (hADSCs) in concave microwells. The hADSCs in spheroids could compensate for the low viability and improve the functional maintenance of hHeps. Co-cultured spheroids aggregated and formed compact spheroidal shapes more rapidly, and with a significantly higher viability than mono-cultured spheroids. The liver-specific functions of co-cultured spheroids were greater, although they contained half the number of hepatocytes as mono-cultured spheroids. Albumin secretion by co-cultured spheroids was 10% higher on day 7, whereas urea secretion was similar, compared with mono-cultured spheroids. A quantitative cytochrome P450 assay showed that the enzymatic activity of co-cultured spheroids cultured for 9 days was 28% higher than that of mono-cultured spheroids. These effects may be due to the transdifferentiation potential and paracrine healing effects of hADSCs on hHeps. These co-cultured spheroids may be useful for creating artificial three-dimensional hepatic tissue constructs and for cell therapy with limited numbers of human hepatocytes.
PLoS ONE 01/2012; 7(12):e50723. · 4.09 Impact Factor
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ABSTRACT: We have developed a size-controllable spheroidal hepatosphere and heterosphere model by mono-culturing of primary hepatocytes and by co-culturing primary hepatocytes and hepatic stellate cells (HSCs). We demonstrated that uniform-sized heterospheres, which self-aggregated from primary hepatocytes and HSCs, formed within concave microwell arrays in a rapid and homogeneous manner. The effect of HSCs was quantitatively and qualitatively investigated during spheroid formation, and HSC played an important role in controlling the organization of the spheroidal aggregates and formation of tight cell-cell contacts. An analysis of the metabolic function showed that heterospheres secreted 30% more albumin than hepatospheres on day 8. In contrast, the urea secretion from heterospheres was similar to that of hepatospheres. A quantitative cytochrome P450 assay showed that the enzymatic activity of heterospheres cultured for 9 days was higher as compared with primary hepatospheres. These size-controllable heterospheres could be mass-produced using concave plate and be useful for creating artificial three-dimensional hepatic tissue constructs and regeneration of failed liver.
Biomaterials 08/2011; 32(32):8087-96. · 7.40 Impact Factor
