Isolated Hepatocytes versus Hepatocyte Spheroids: In Vitro Culture of Rat Hepatocytes

Abstract

The use of hepatocytes that express liver-specific functions to develop an artificial liver is promising. Unfortunately, the loss of specialized liver functions (dedifferentiation) is still a major problem. Different techniques, such as collagen entrapment, spherical multicellular aggregates (spheroids), and coculture of hepatocytes with extracellular matrix, have been used to improve the performance of hepatocytes in culture. The aim of this study was to compare two different models of hepatocyte isolation in culture: isolated hepatocytes (G1) and hepatocyte spheroids (60% hepatocytes, 40% nonparenchymal cells, and extracellular matrix) (G2). To test functional activity of hepatocytes, both synthetic and metabolic, production of albumin and benzodiazepine transformation into metabolites was tested. G2 showed a high albumin secretion, while a decrease after 15 days of culture in G1 was noted. Diazepam metabolites were higher in G2 than in G1 in all samples, but had statistical significance at days 14 and 21 (p < 0.01). The glycogen content, after 30 days of culture, was very low in G1 (14.2 ± 4.4%), while in G2 it was 72.1 ± 2.6% (p < 0.01). Our study confirms the effectiveness of a culture technique with extracellular matrix and nonparenchymal cells. Maintenance of a prolonged functional activity has been related to restoration of cell polarity and close cell-to-cell contact. We showed that isolated hepatocytes maintain their functional activity for a period significantly reduced, when compared to the hepatocyte spheroids. We confirmed the role of extracellular matrix as a crucial component to promote hepatocyte homeostasis, and the close link between cellular architecture and tissuespecific functions.

E-Cadherin Protects Primary Hepatocyte Spheroids from Cell Death by a Caspase-Independent Mechanism

“…Under these conditions, freshly isolated rat hepatocytes aggregate spontaneously to form spheroids of 60–120 μm diameter by 24 h and viability exceeding 95%. As previously shown by others (1), differentiated hepatocyte functions were better maintained in spheroid culture. However, cell loss became problematic over extended culturing periods.…”

Human hepatocyte transplantation: state of the art

“…The mainstay of management is with daily phototherapy though the inconvenience and risk of hyperbilirubinaemia during intercurrent illness means a permanent replacement of function with liver transplantation is appealing. Hepatocyte transplantation has been successfully used as an alternative to whole organ transplantation with a transient decrease in bilirubin for up to 11 months [4, 28–33].…”

The modern creation technologies of implanted bioartificial liver

“…Ambrosino G. с соавт. [33] показали, что изолированные гепатоциты поддерживали функциональную активность в течение значительно меньшего периода времени по сравнению со сфероидами гепатоцитов. Авторы получили изолированные гепатоциты и гепатоциты в виде сфероидов (60% гепатоцитов, 40% непаренхиматозных клеток и экстрацеллюлярный матрикс) и подтвердили роль экстрацеллюлярного матрикса как критического компонента для обеспечения гомеостаза гепатоцитов и роль тесной связи между клеточной архитектурой и тканеспецифическими функциями [33].…”

“…Авторы получили изолированные гепатоциты и гепатоциты в виде сфероидов (60% гепатоцитов, 40% непаренхиматозных клеток и экстрацеллюлярный матрикс) и подтвердили роль экстрацеллюлярного матрикса как критического компонента для обеспечения гомеостаза гепатоцитов и роль тесной связи между клеточной архитектурой и тканеспецифическими функциями [33].…”