Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution

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(DMSOUW) 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(r 2 = 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.

The potential use of extended criteria donors and eligible recipients in liver transplantation for unresectable colorectal liver metastases in Central Sweden

Background:Unresectable colorectal liver metastases(CRLM)is a condition with poor prognosis.A recent treatment alternative improving survival in patients with unresectable CRLM,has emerged with the introduction of liver transplantation(LT),yet not uncontroversial with the current organ shortage.This study aimed to retrospectively investigate the potential of declined donors with acceptable risk as liver graft donors and patients with unresectable CRLM as potential recipients.Methods:All declined donors in central Sweden and all patients with CRLM discussed at multidisciplinary team conference at Karolinska University Hospital,January 2013-October 2018,were identified.Donors were classified according to the European Committee Guide to the quality and safety of organs for transplantation and potential recipients were evaluated by selection criteria,based on studies on the Norwegian Secondary Cancer study database.Results:Out of 1,462 evaluated potential donors,62(2.7 pmp)donors were identified,corresponding to 6-18%of the utilized donor pool.Out of 1,008 included patients with CRLM,25(2.1 pmp)potential recipients were recognized.Eligibility for LT and left-sided colon cancer were favorable prognostic factors.Conclusions:Today’s donor pool could increase with the use of extended criteria donors,which is sufficient and display an acceptable risk-benefit ratio for patients with unresectable CRLM.With current selection criteria a small subset of patients with unresectable CRLM are eligible recipients.This subset of patients has a better survival compared to patients ineligible for LT.