Characterization of human hepatocytes isolated from non-transplantable livers.
BACKGROUND
The successful use of hepatocytes depends on a reliable demonstration of the functional and morphological integrity of isolated cells. Herein we investigated whether the isolation and cryopreservation of primary human hepatocytes can compromise cell viability and liver-specific characteristics.


METHODS
Hepatocytes were isolated from encapsulated human liver segments by a modified 2-step perfusion technique. Isolated cells were Percoll-purified, cryopreserved, and stored in liquid nitrogen for 1-12 months. For rapid assessment of fresh and cryopreserve/thawed hepatocyte yield and viability, the cells were stained with trypan blue or labeled with fluorochromes. For immunocytochemical analysis, the cells were labeled with monoclonal antibodies for the presence of the following antigens and chemokines: CD3, CD45Ro, CD45Ra, CD34, CD68, CD90, CD95, CD20, HLA-DR, Ki67, PCNA, Bcl-2, p53, CXCR3, CXCR4, and SDF-1. The cells were tested for several specific functions, such as ureagenesis, energy status, MTT activity, lactate dehydrogenase leakage, and total CYP450 content.


RESULTS
Assessment of both freshly isolated (Percoll-purified) and cryopreserved/thawed hepatocytes revealed a low constitutive level of contamination by non-parenchymal cells compared with crude (unpurified) preparations and tissue sections. All viable hepatocytes showed intact morphology and retained CYP450 protein, energy status, and urea synthesis.


CONCLUSIONS
Modifications in hepatocyte preparations, such as depletion of dead, damaged, and nonparenchymal cells, improves cell purity, which can be adapted to further evaluation of hepatocyte immunogenicity. These data illustrate the importance and feasibility of human hepatocyte banking.
Łaba
Anna
A
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland. laba@iitd.pan.wroc.pl
Ostrowska
Alina
A
Patrzałek
Dariusz
D
Paradowski
Leszek
L
Lange
Andrzej
A
D000255:Adenosine Triphosphate / Q000032:analysis; D000328:Adult; D002469:Cell Separation / Q000379:methods*; D015925:Cryopreservation; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis; D005260:Female; D022781:Hepatocytes / Q000737:chemistry / Q000166:cytology*; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D054367:Receptors, CXCR3; D019718:Receptors, CXCR4 / Q000032:analysis; D019707:Receptors, Chemokine / Q000032:analysis
C515187:CXCR3 protein, human; D054367:Receptors, CXCR3; D019718:Receptors, CXCR4; D019707:Receptors, Chemokine; D000255:Adenosine Triphosphate; D003577:Cytochrome P-450 Enzyme System