A novel formulation of oxygen‐carrying matrix enhances liver‐specific function of cultured hepatocytes

Abstract

Oxygen is an important component of the cellular microenvironment, mediating cell survival, differentiation, and function. Oxygen supply is a limiting factor during culture of highly metabolic cells such as hepatocytes. Here we present a simple formulation of a fluorocarbon-based oxygen carrier embedded in collagen gel that increases oxygen concentration in culture 6-fold. Rat hepatocytes cultured on oxygen carrier-collagen showed a significant increase in viability and function. Cytochrome P450IA1 activity was increased by 140% in serum-free cultures and by 820% in serum-containing cultures. The significantly higher hepatocellular function on oxygen carrier-collagen matrix persisted and increased during long-term culture. Long-term albumin secretion was increased by 350% in serum-free cultures and by 166% in serum-containing culture. Long-term urea secretion was increased by 79% in serum-free cultures and by 76% in serum-containing cultures. We conclude that oxygen supply may limit hepatocyte function in vitro. This limitation can be overcome by addition of an oxygen carrier to the extracellular matrix. Culture of hepatocytes on oxygen-carrying matrix mimics the oxygen-rich environment of the liver and provides a simple method for enhanced long-term function.

Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance

“…However, as the cells adapt to their new microenvironment, oxygen uptake rates drop to 0.4 nmol/s/10 6 cells during long-term culture (17,18). Not surprisingly, 0.4 nmol/s/10 6 cells is also the upper limit of oxygen diffusion under atmospheric oxygen, but significantly less than hepatocyte demand during seeding (16). Metabolic flux models also suggest that hepatocytes in culture attempt to maximize their oxygen uptake (41).…”

“…A critical aspect of the microenvironment which is dramatically different between in vivo and in vitro is oxygen supply (15). In vivo a mixture of arterial and venous blood continuously supply over 2,000 nmol/mL of oxygen to hepatocytes, while in vitro oxygen's low solubility in culture media offers less than 200 nmol/mL to the cells (7,16). While this has traditionally limited hepatocytes to subconfluent cultures (15), oxygen supply becomes an even greater concern during the initial phase of cell attachment when oxygen uptake rates are 300% greater than normal (17,18).…”

“…Our recent development of an oxygen-carrying matrix allowed us to identify a negative effect of serum on oxygen-enhanced metabolism (16). As serum has been previously shown to cause loss of hepatocyte polarity and gene expression during the onset of culture (23,24), it suggests that a serum-free, oxygen-rich microenvironment would minimize adaptation stress and allow for high levels of metabolic activity from the onset of culture.…”

The modern creation technologies of implanted bioartificial liver

“…Снабжение кислородом является лимитирующим фактором при культивировании активно метаболизирующих клеток, таких как гепатоциты [9][10][11]. Nahmias и соавт.…”

“…Секреция мочевины увеличивалась на 79% СОЗДАНИЕ ИМПЛАНТИРУЕМОЙ БИОИСКУССТВЕННОЙ ПЕЧЕНИ 426 в бессывороточных культурах и на 76% в культурах с сывороткой. Культивирование гепатоцитов на матриксе, обогащенном кислородом, имитирует реальное микроокружение печени, богатое кислородом, и обеспечивает простой метод для получения более полноценной и долгосрочной функции гепатоцитов [10].Kim S. с соавт.[12] году успешно культивировали гепатоциты крысы в проточных условиях на 3х-мерном матриксе из сополимера поли-молочной и поли-гликолевой кислот с внутренней сетью каналов. Авторы использовали среду William's с пируватом натрия для выделения гепатоцитов и среду Dulbecco с 10% сывороткой, дексаметазоном и инсулином для кокультивирования гепатоцитов и непаренхиматозных клеток печени.…”