An optimised method for cryopreservation of human hepatocytes.

Institute of Liver Studies, King's College London School of Medicine London, UK.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2009; 481:25-34. DOI: 10.1007/978-1-59745-201-4_3
Source: PubMed


Successful cryopreservation of hepatocytes is essential for their use in hepatocyte transplantation. Cryopreservation allows hepatocytes to be available for emergency treatment of acute liver failure and also for planned treatment of liver-based metabolic disorders. In addition, cryopreservation of human hepatocytes can facilitate their use in metabolism and toxicity studies. Cryopreservation can adversely affect the viability and function, especially reduce the attachment efficiency, of hepatocytes on thawing.The cryopreservation process can be divided into steps so that improvements can be made on the 'standard' protocols that are followed in some laboratories. These steps are as follows: pre-incubation of cells; freezing solution, cryoprotectants and cytoprotectants; freezing process; storage; thawing; post-thawing culture. This chapter presents an optimised protocol for cryopreservation of human hepatocytes as developed at King's College Hospital.

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    • "Live-cell bio-banks utilize heterogeneous cell populations whose functional properties need to be preserved. For example, peripheral blood mononuclear cells must maintain their characteristic immune profile, hepatocytes must preserve their metabolic activity [22], and embryonic stem cells must retain their pluripotency after cryopreservation [23]. Hence, successful cryopreservation relies on the ability to observe and retrieve a specific functioning cell or cell group. "
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    ABSTRACT: Cryopreservation is the only widely applicable method of storing vital cells for nearly unlimited periods of time. Successful cryopreservation is essential for reproductive medicine, stem cell research, cord blood storage and related biomedical areas. The methods currently used to retrieve a specific cell or a group of individual cells with specific biological properties after cryopreservation are quite complicated and inefficient. The present study suggests a new approach in cryopreservation, utilizing the Individual Cell-based Cryo-Chip (i3C). The i3C is made of materials having appropriate durability for cryopreservation conditions. The core of this approach is an array of picowells, each picowell designed to maintain an individual cell during the severe conditions of the freezing--thawing cycle and accompanying treatments. More than 97% of cells were found to retain their position in the picowells throughout the entire freezing--thawing cycle and medium exchange. Thus the comparison between pre-freezing and post-thawing data can be achieved at an individual cell resolution. The intactness of cells undergoing slow freezing and thawing, while residing in the i3C, was found to be similar to that obtained with micro-vials. However, in a fast freezing protocol, the i3C was found to be far superior. The results of the present study offer new opportunities for cryopreservation. Using the present methodology, the cryopreservation of individual identifiable cells, and their observation and retrieval, at an individual cell resolution become possible for the first time. This approach facilitates the correlation between cell characteristics before and after the freezing--thawing cycle. Thus, it is expected to significantly enhance current cryopreservation procedures for successful regenerative and reproductive medicine.
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