Optimization of the cryopreservation and thawing protocol for human hepatocytes for use in cell transplantation

Abstract

Cryopreservation of human hepatocytes is important for their use in hepatocyte transplantation. On thawing, cryopreserved hepatocytes often have reduced viability and metabolic function in comparison with fresh cells. The aim of this study was to modify the different steps in the standard cryopreservation procedure in an attempt to improve the overall outcome. Human hepatocytes with a viability of 69% 6 SD 16% were isolated from donor livers with a collagenase perfusion technique. Different cell densities, concentrations, rates, and methods of addition of dimethyl sulfoxide were tested for the freezing solution. Modified controlled-rate freezer programs were tested to obtain a linear decrease in the temperature. Once they were frozen, the storage time and thawing method for hepatocytes were investigated. The effects on thawed cell viability and attachment, lactate dehydrogenase release, cytochrome P450 1A1/2 activity, and albumin synthesis were determined. The results were used to produce an improved cryopreservation protocol suitable for good manufacturing practice conditions. With a cell density of 10 7 cells/mL in University of Wisconsin solution containing 300 mM glucose, 10% (vol/vol) dimethyl sulfoxide was added dropwise over 5 minutes, and was immediately frozen. Thawing was done rapidly at 37 C, and dilution was performed with Eagle's minimum essential medium containing 300 mM glucose and 4% human serum albumin. Hepatocytes could be stored at À140 C without significant further loss of function for up to 3 years. With this protocol, hepatocytes had a viability of 52% 6 9%, an attachment efficiency of 48% 6 8%, and lactate dehydrogenase leakage of 17% 6 4%. This protocol is currently in use to cryopreserve hepatocytes for use in cell transplantation at our center. Liver Cryopreservation of human hepatocytes is essential for their use in hepatocyte transplantation. Successful cryopreservation allows hepatocytes to be available for the emergency treatment of acute liver failure and also for planned and repeated treatments with cells from the same donor for liver-based metabolic disorders. However, it is well known that cryopreservation affects the viability and metabolic function of hepatocytes on thawing, with the result that the thawed cells are often not suitable for clinical use.A considerable number of human hepatocyte cryopreservation protocols have been reported for experimental use.

Human Hepatocyte Transplantation in Patients with Hepatic Failure Awaiting a Graft

“…The response of HT is likely to rely on many different factors, among them, the nature of the disease, the number of cells required/administered, their functional performance, and the cells' ability to engraft and survive in the host organ [10,19,20,24,26,27]. One major problem is how to assess the clinical efficacy of HT; we assessed, as other autors, the efficacy mainly based on clinical criteria, which proves more difficult to objectify the analytical data, primarily ammonia levels [6,7,8,9,14,17].…”

Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

“…The freezing protocol was chosen based on results of a preliminary study. The published protocols by Terry et al 24 for cryopreservation of human hepatocytes and the protocol for freezing alginate-encapsulated liver cell spheroids by Massie et al 25 were compared. These 2 protocols are multistep with slow cooling and contain an important holding step to allow samples to reach equilibrium followed by a shock cooling step (from À8 C toÀ28 C) to control nucleation of ice.…”

“…Following storage at À140 C for 2 wk, isolated cells and microbeads were rapidly thawed in a 37 C water bath 24,28 using ice-cold Eagle's minimum essential medium (EMEM) (EMEM; Lonza, Verviers, Belgium) as thawing media. Similarly, cryopreserved HMBs were thawed with 2% (v/v) HSA added in EMEM.…”

Hepatocyte Transplantation

“…It must be noted that the quality of the isolated HCs will depend on the quality of the liver tissue from which they were isolated. It is best to transplant HCs fresh, but if no patient is waiting, these HCs can be cryopreserved using optimized protocols, 24 though there will be some loss of HC metabolic function on thawing.…”