Use of indocyanine green for functional assessment of human hepatocytes for transplantation.
OBJECTIVE
Hepatocyte transplantation is a promising alternative to liver transplantation in children with liver metabolic disorders and acute liver failure. Currently, it is difficult to assess rapidly hepatocyte function before transplantation. The aim of this study was to investigate whether the uptake and release of indocyanine green (ICG) by hepatocytes could be used.


METHODS
Human hepatocytes (10(6) cells) isolated from unused donor livers were incubated at 37°C for 30 minutes with ICG (0-2mg/mL) in both cell suspension and on collagen-coated culture plates. Cells were then incubated in medium without ICG for 3 hours with supernatants collected at 1, 2 and 3 hours for measurement of ICG release. Cell viability was determined by trypan blue exclusion, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (mitochondrial dehydrogenase activity) and sulforhodamine B (SRB) assay (cell attachment). HepG2 cells were also used.


RESULTS
ICG was taken up and secreted by hepatocytes with the release reaching a plateau level soon after 1 hour. Concentrations of ICG > 1.0mg/mL had toxic effects on hepatocytes. Hepatocytes incubated with 1.0mg/mL ICG had higher mitochondrial dehydrogenase activity compared to 0.5mg/mL ICG or control cells (0.025 ± 0.0004 OD unit vs. 0.019 ± 0.0008 OD unit or 0.020 ± 0.002 OD unit, p<0.05). Incubation of HepG2 cells with ICG reduced albumin production (98.9 ± 0.02 ng/mL, 66.6 ± 0.05 ng/mL and 39.1 ± 0.4 ng/mL for control cells, and 0.5mg/mL and 1.0mg/mL ICG, respectively), and decreased [(3)H]-thymidine incorporation in a dose-dependent manner. Addition of taurine (20mM) to plated hepatocytes gave greater release of ICG and hepatocyte attachment compared to controls, at all ICG concentrations (SRB 1.360 ± 0.083 optical density units vs. 0.908 ± 0.159 optical density units, p=0.011 at 1.0mg/mL).


CONCLUSIONS
With further refinement, ICG could be used to develop a rapid assay for assessment of the function of isolated human hepatocytes.
Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan.
Cheng-Maw
CM
Ho
Anil
A
Dhawan
Robin D
RD
Hughes
Sharon C
SC
Lehec
Juliana
J
Puppi
Christina
C
Philippeos
Po-Huang
PH
Lee
Ragai R
RR
Mitry
D000328:Adult; D000368:Aged; D000418:Albumins / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents / Q000493:pharmacokinetics / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D005260:Female; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism / Q000637:transplantation*; D006801:Humans; D007208:Indocyanine Green / Q000493:pharmacokinetics / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D008930:Mitochondria, Liver / Q000378:metabolism
D000418:Albumins; D004396:Coloring Agents; D004247:DNA; D007208:Indocyanine Green