Abstract
Primary hepatocytes undergo phenotypic dedifferentiation upon isolation from liver that typically includes down regulation of uptake transporters and up regulation of efflux transporters. Culturing cells between layers of collagen in a three-dimensional (3D) “sandwich” is reported to restore hepatic phenotype. This report examines how 3D culturing affects accumulation of fluorophores, the cytotoxic response to bile acids and drugs, and whether cell to cell differences in fluorescent anion accumulation correlate with differences in cytotoxicity. Hepatocytes were found to accumulate fluorescent bile acid (FBA) at significantly higher levels than the related fluorophores, carboxyfluorescein diacetate, (4.4-fold), carboxyfluorescein succinimidyl ester (4.8-fold), and fluorescein (30-fold). In 2D culture, FBA accumulation decreased to background levels by 32 h, Hoechst nuclear accumulation strongly decreased, and nuclear diameter increased, indicative of an efflux phenotype. In 3D culture, FBA accumulation was maintained through 168 h but at 1/3 the original intensity. Cell to cell differences in accumulated FBA did not correlate with levels of liver zonal markers L-FBAP (zone 1) or glutamine synthetase (zone 3). Cytotoxic response to hydrophobic bile acids, acetaminophen, and phalloidin was maintained in 3D culture, and cells with higher FBA accumulation showed 12–18% higher toxicity than the total population toward hydrophobic bile acids (P < 0.05). Long-term imaging showed oscillations in the accumulation of FBA over periods of hours. Overall, the studies suggest that high accumulation of FBA can indicate the sensitivity of cultured hepatocytes to hydrophobic bile acids and other toxins.
| Original language | English (US) |
|---|---|
| Article number | e12198 |
| Journal | Physiological Reports |
| Volume | 2 |
| Issue number | 12 |
| DOIs | |
| State | Published - 2014 |
Fingerprint
Keywords
- Collagen sandwich
- Fluorescent bile acid (FBA)
- Hepatocyte culture
ASJC Scopus subject areas
- Physiology (medical)
- Physiology
Cite this
Hepatocytes maintain greater fluorescent bile acid accumulation and greater sensitivity to drug-induced cell death in three-dimensional matrix culture. / Murray, John W.; Han, Dennis; Wolkoff, Allan W.
In: Physiological Reports, Vol. 2, No. 12, e12198, 2014.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Hepatocytes maintain greater fluorescent bile acid accumulation and greater sensitivity to drug-induced cell death in three-dimensional matrix culture
AU - Murray, John W.
AU - Han, Dennis
AU - Wolkoff, Allan W.
PY - 2014
Y1 - 2014
N2 - Primary hepatocytes undergo phenotypic dedifferentiation upon isolation from liver that typically includes down regulation of uptake transporters and up regulation of efflux transporters. Culturing cells between layers of collagen in a three-dimensional (3D) “sandwich” is reported to restore hepatic phenotype. This report examines how 3D culturing affects accumulation of fluorophores, the cytotoxic response to bile acids and drugs, and whether cell to cell differences in fluorescent anion accumulation correlate with differences in cytotoxicity. Hepatocytes were found to accumulate fluorescent bile acid (FBA) at significantly higher levels than the related fluorophores, carboxyfluorescein diacetate, (4.4-fold), carboxyfluorescein succinimidyl ester (4.8-fold), and fluorescein (30-fold). In 2D culture, FBA accumulation decreased to background levels by 32 h, Hoechst nuclear accumulation strongly decreased, and nuclear diameter increased, indicative of an efflux phenotype. In 3D culture, FBA accumulation was maintained through 168 h but at 1/3 the original intensity. Cell to cell differences in accumulated FBA did not correlate with levels of liver zonal markers L-FBAP (zone 1) or glutamine synthetase (zone 3). Cytotoxic response to hydrophobic bile acids, acetaminophen, and phalloidin was maintained in 3D culture, and cells with higher FBA accumulation showed 12–18% higher toxicity than the total population toward hydrophobic bile acids (P < 0.05). Long-term imaging showed oscillations in the accumulation of FBA over periods of hours. Overall, the studies suggest that high accumulation of FBA can indicate the sensitivity of cultured hepatocytes to hydrophobic bile acids and other toxins.
AB - Primary hepatocytes undergo phenotypic dedifferentiation upon isolation from liver that typically includes down regulation of uptake transporters and up regulation of efflux transporters. Culturing cells between layers of collagen in a three-dimensional (3D) “sandwich” is reported to restore hepatic phenotype. This report examines how 3D culturing affects accumulation of fluorophores, the cytotoxic response to bile acids and drugs, and whether cell to cell differences in fluorescent anion accumulation correlate with differences in cytotoxicity. Hepatocytes were found to accumulate fluorescent bile acid (FBA) at significantly higher levels than the related fluorophores, carboxyfluorescein diacetate, (4.4-fold), carboxyfluorescein succinimidyl ester (4.8-fold), and fluorescein (30-fold). In 2D culture, FBA accumulation decreased to background levels by 32 h, Hoechst nuclear accumulation strongly decreased, and nuclear diameter increased, indicative of an efflux phenotype. In 3D culture, FBA accumulation was maintained through 168 h but at 1/3 the original intensity. Cell to cell differences in accumulated FBA did not correlate with levels of liver zonal markers L-FBAP (zone 1) or glutamine synthetase (zone 3). Cytotoxic response to hydrophobic bile acids, acetaminophen, and phalloidin was maintained in 3D culture, and cells with higher FBA accumulation showed 12–18% higher toxicity than the total population toward hydrophobic bile acids (P < 0.05). Long-term imaging showed oscillations in the accumulation of FBA over periods of hours. Overall, the studies suggest that high accumulation of FBA can indicate the sensitivity of cultured hepatocytes to hydrophobic bile acids and other toxins.
KW - Collagen sandwich
KW - Fluorescent bile acid (FBA)
KW - Hepatocyte culture
UR - http://www.scopus.com/inward/record.url?scp=85003550850&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85003550850&partnerID=8YFLogxK
U2 - 10.14814/phy2.12198
DO - 10.14814/phy2.12198
M3 - Article
AN - SCOPUS:85003550850
VL - 2
JO - Physiological Reports
JF - Physiological Reports
SN - 2051-817X
IS - 12
M1 - e12198
ER -