The Viability and Function of Cryopreserved Hepatocyte Spheroids with Different Cryopreservation Solutions

Park, Jae Berm; Lee, Kwang Woong; Kim, Sung Joo; Lee, Suk Koo; Park, Je Hoon; Woo, Don Hee; Joh, Jae Won

Abstract

PURPOSE
The bioartificial liver via extracorporeal circulation and the hepatocyte transplantation have been studied due to donor organ shortage. Hepatocyte spheroids, which are tightly packed multicellular aggregates, showed enhanced liver specific activities. The authors have studied the bioartificial liver system using hepatocyte spheroids. For the effective application of this system, the effective cryopreservation technique is necessary. In this study, the aim was to evaluate the viability and function of cryopreserved hepatocyte spheroids with different cryopreservation solutions and to elucidate the efficiency of cryopreservation. METHODS: Hepatocytes were isolated Sprague-Dawley rat. The hepatocyte spheroids were formed with 24 hours by rotational culturing. The hepatocyte spheroids were frozen in different cryopreservation solutions (UW solution, William E media, FBS and mixture) by programmed linear freezer, preserved in liquid nitrogen tank for 24 hours and cultured for 4 days after thawing. For the viabilities of each hepatocyte spheroids, the MTT assay was made and for their hepatocyte specific functions, ammonia clearance, urea nitrogen synthesis and albumin secretion of the spheroids were evaluated. RESULTS: The viabilities of the cyropreserved hepatocyte spheroids after culturing for 4 hours following thawing were 64.8+/-10.2, 33.2+/-9.7, 69.3+/-8.7 and 48.4+/-15.5% in the UW, WE, FBS and MIX media, respectively. The ammonia clearance of the spheroids cyropreserved in the UW solution was 0.93+/-0.13 mM/well/day, which was not significantly different from that of the freshly cultured spheroids. With regard to the urea nitrogen synthesis, the cryopreserved spheroids in the UW, FBS and MIX solutions were not significantly different from the freshly cultured spheroids. The amount of albumin secretion of the cryopreserved spheroids in the UW solution was significantly higher than those of cryopreserved spheroids in the other solutions. CONCLUSION: With regard to the viability and function, the hepatocyte spheroids cryopreserved in the UW solution were not significantly different from the freshly cultured spheroids, which were superior to the other cryopreserved spheroids. Further studies relating to the optimal culture and cryopreservation environments, such as freezing rate or cryoprotectant from damage during freezing, are needed.