Controlled formation of heterotypic hepatic micro-organoids in anisotropic hydrogel microfibers for long-term preservation of liver-specific functions

We have developed a hydrogel-based cell cultivation platform for forming 3D restiform hepatic micro-organoids consisting of primary rat hepatocytes and feeder cells (Swiss 3T3 cells). Sodium alginate solutions containing hepatocytes/3T3 cells were continuously introduced into a microfluidic channel to produce cell-incorporating anisotropic Ba-alginate hydrogel microfibers, where hepatocytes at the center were closely sandwiched by 3T3 cells. Hydrogel fiber-based cultivation under high oxygen tension enabled the formation of heterotypic micro-organoids with a length of up to 1 mm and a diameter of similar to 50 mu m, mimicking the hepatic cord structures found in the liver, while maintaining a high hepatocyte viability (similar to 80%) over 30 days. Long-term observation of up to 90 days revealed a significant enhancement of hepatic functions because of heterotypic and homotypic cell cell interactions, including albumin secretion and urea synthesis as well as expression of hepatocyte-specific genes, compared with conventional monolayer culture and single cultivation in the hydrogel fibers. The encapsulated hepatic constructs were recovered as scaffold-free micro-organoids by enzymatically digesting the hydrogel matrices using alginate lyase. This technique for creating heterotypic micro-organoids with precisely ordered multiple cell types will be useful for the development of a new liver tissue engineering approach and may be applicable to the fabrication of extracorporeal bioartificial liver (BAL) devices and assessment tools for drug development and testing. (C) 2012 Elsevier Ltd. All rights reserved.