Plated Hepatocyte Co-Culture for Long-Term Liver Function Testing
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Solution Overview
Problem
Existing 2D in vitro culture systems for primary human hepatocytes face challenges with low plateability, functional stability, and culture longevity, particularly for conditions like non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), limiting their suitability for long-term drug metabolism and toxicity testing.
Innovation Solution
Co-culturing human hepatocytes with endothelial cells and fibroblasts forms hepatocyte clusters on a surface, enhancing plateability and maintaining functional stability and culture longevity, with at least 70% of hepatocytes in direct contact and covering at least 50% of the surface, and remaining viable for up to 42 days.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If primary human hepatocytes are cultured in conventional 2D monolayer systems, then the culture setup is simple, but plateability is low and culture longevity is limited
Solution Approach 1:
The patent combines hepatocytes with feeder cells (endothelial cells and fibroblasts) in a co-culture system. This merging of cell types creates a more complex cellular environment that supports hepatocyte attachment and longevity, resolving the contradiction between simple culture setup and reliable plateability
Solution Approach 2:
The invention uses a composite cell culture system comprising multiple cell types (hepatocytes, endothelial cells, fibroblasts) rather than a single cell type. This composite approach enhances plateability and culture stability while accepting increased system complexity
2Duration of action of stationary object
If hepatocytes are maintained for long-term culture, then culture longevity is extended, but functional stability deteriorates
Solution Approach 1:
The feeder cells in the co-culture system provide self-service by secreting growth factors and cytokines that automatically support hepatocyte survival and function over time. This creates a self-sustaining environment that maintains functional stability during long-term culture without external intervention
Solution Approach 2:
The co-culture system establishes continuous beneficial interactions between hepatocytes and feeder cells, where feeder cells continuously provide supportive signals and hepatocytes maintain their functional phenotype. This continuous mutual support resolves the contradiction between extended culture duration and maintained functional stability
3Area of stationary object
If more hepatocytes are plated to achieve high coverage, then surface coverage increases, but plateability decreases
Solution Approach 1:
Feeder cells act as intermediaries between the culture surface and hepatocytes. These intermediary cells create a favorable microenvironment that facilitates hepatocyte attachment and spreading, enabling high surface coverage while maintaining high plateability that would otherwise be contradictory
Data Source
AI summary
The present invention provides a product comprising plated human hepatocytes on a surface and at least some of the plated hepatocytes are in one or more hepatocyte clusters on feeder cells, which are attached to the surface. A method of preparing plated human hepatocytes is also provided. The preparation method comprises applying human hepatocytes to a surface in the presence of feeder cells, co-culturing the applied hepatocytes with the feeder cells, and forming one or more hepatocyte clusters by the co-cultured hepatocytes on the feeder cells, which are attached to the surface. The plated hepatocytes may be used for various purposes, including the preparation of a hepatitis B virus (HBV) infected hepatocyte culture model and drug testing.