Brief Article
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World J Hepatol. May 27, 2012; 4(5): 176-183
Published online May 27, 2012. doi: 10.4254/wjh.v4.i5.176
Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution
Mohammed Saliem, Frida Holm, Rosita Bergström Tengzelius, Carl Jorns, Lisa-Mari Nilsson, Bo-Göran Ericzon, Ewa Ellis, Outi Hovatta
Mohammed Saliem, Frida Holm, Rosita Bergström Tengzelius, Outi Hovatta, Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institute, 141 86 Stockholm, Sweden
Carl Jorns, Bo-Göran Ericzon, Ewa Ellis, Division of Transplantation Surgery, Department of Clinical Science, Intervention and Technology, Karolinska Institute, 141 86 Stockholm, Sweden
Lisa-Mari Nilsson, Division of Pediatrics, Department of Clinical Science, Intervention and Technology, Karolinska Institute, 141 86 Stockholm, Sweden
Author contributions: The last two authors, Ellis E and Hovatta O, contributed equally to this work; Saliem M, Holm F, Ericzon BG, Ellis E and Hovatta O designed the study; Saliem M, Jorns C, Nilsson LM and Ellis E performed the study; Saliem M, Bergström R and Ellis E performed the data analysis; and Saliem M, Holm F, Bergström Tengzelius R, Ericzon BG, Ellis E and Hovatta O wrote the manuscript.
Supported by Grants from VINNMER, Lundin foundation, R&D Funds from Stockholm County and Karolinska Institutet (ALF), and the Swedish Research Council
Correspondence to: Ewa Ellis, PhD, Division of Transplantation Surgery, Department of Clinical Science, Intervention and Technology, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden. ewa.ellis@ki.se
Telephone: +46-8-58580086 Fax: +46-8-7743191
Received: March 29, 2011
Revised: September 19, 2011
Accepted: April 25, 2012
Published online: May 27, 2012
Abstract

AIM: To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.

METHODS: The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes. Despite several hepatocyte cryopreservation protocols being available, improvements are urgently needed. We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups. Using the polystyrene box freezing, we compared two xeno-free freezing solutions for freezing of primary human hepatocytes: a new medium (STEM-CELLBANKER, CB), which contains dimethylsulphoxide (DMSO) and anhydrous dextrose, both permeating and non-permeating cryoprotectants, and the frequently used DMSO - University of Wisconsin (DMSO-UW) medium. The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation. The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms (CYPs): CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A7.

RESULTS: The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol (P < 0.01). There was no significant difference in viability estimation between both the trypan blue (TB) and the Live-Dead Assay methods. There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols (r2 = 0.69) using the TB method. However, due to high within-group variability in the activities of the major CYPs, any statistical between-group differences were precluded. Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability. Thus, it may be a better alternative to the standard DMSO-UW protocol. Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.

CONCLUSION: The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.

Keywords: Human hepatocytes; Viability; Cytochrome P540; Dimethylsulphoxide; Cryoprotectant; Cryopreservation