Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

Abstract

Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimized protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300 mM) glucose and CryoStor CS10 showed better postthawing cell viability, attachment, and hepatocyte functions than with histidine-tryptophan-ketoglutarate/10% DMSO/5% glucose and Bambanker. The 2 freezing solutions that gave better results were studied with human and rat hepatocytes microbeads. Similar effects on cryopreserved microbead morphology (external and ultrastructural), viability, and hepatocyte-functions post thawing were observed over 7 d in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone [ZVAD]), an antioxidant (desferoxamine [DFO]), and a buffering and mechanical protectant (human serum albumin [HSA]) on RMBs. ZVAD (60 mM) had a beneficial effect on cell viability that was greater than with DFO (1 mM), HSA (2%), and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes and a lower degree of cell apoptosis were observed with all 3 cytoprotectants, with ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the 3 cytoprotectant groups than with fresh microbeads. In conclusion, developing an optimized cryopreservation protocol by adding cytoprotectants such as ZVAD could improve the outcome of cryopreserved hepatocyte microbeads for future clinical use.

Cell therapy for advanced liver diseases: Repair or rebuild

“…45 The use of apoptosis inhibitors is also effective in improving thawed hepatocyte viability and function of alginate-encapsulated and cryopreserved hepatocyte microbeads. 46 The microenvironment in which cells are transplanted is a critical consideration in the success of cell therapy. Cells enter the liver via sinusoids where they integrate with the parenchyma.…”

Liver Bioengineering: Promise, Pitfalls, and Hurdles to Overcome

“…2b). However, it was shown that even cryopreservation has a considerable effect on the viability and metabolic function of these cells due to cells being subjected to damaging conditions during both freezing and thawing steps [47][48][49][50]. To increase cryopreserved cell viability, Jitraruch et al developed an optimized protocol for cryopreservation of hepatocyte microbeads using modified freezing solutions [47].…”

“…Moreover, allogenic hepatocyte transplantation should be still followed by an immunosuppressive therapy used for OLT [44,74,76]. To overcome these limitations, cells can be transplanted within a biodegradable scaffold, such as microcapsules and microbeads, to provide a microenvironment suitable for cell attachment and also protect cells from host's immune system [47,77]. The fact that hepatocytes are typically harvested from livers not suitable for transplantation makes quantity and quality of cells obtained highly variable [78].…”

Advances in the slow freezing cryopreservation of microencapsulated cells

“…Those observations are insufficient to determine capsule stiffness and pore size, and assure the capsules without the antioxidants, their efficiency could be questioned [12]. On the other hand, anti-apoptotic factor effects in CPA solution were investigated with hepatocytes reducing apoptosis significantly in the first 24 hours after thawing [81]. In this regard, cell death and apoptosis behavior within microcapsules have not been carefully studied after cryopreservation.…”

“…The only method to preserve cell-based systems or products for long periods is with the use of low temperatures. Thus, the efforts to cryopreserve cell-based products made of natural polymers (e.g., agarose, hyaluronic acid, fibrin or collagen) or synthetic polymers (e.g., PEG) have linearly grown with its reflection in more publications in last decades[22,30,47,79,81]. Alginate has been one of the most often used polysaccharides to form cellularized bioscaffolds due to its potential as sustained drug delivery or cell delivery systems for the treatment of different diseases (e.g., diabetes, hepatic diseases, neurodegenerative or cancer among others)[82][83][84][85].…”