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Microencapsule technique protects hepatocytes from cryoinjury
Author(s) -
Kusano Tomokazu,
Aoki Takeshi,
Yasuda Daisuke,
Matsumoto Shuichiro,
Jin Zhenghao,
Nishino Nobukazu,
Hayashi Ken,
Odaira Masanori,
Yamada Kousuke,
Koizumi Tomotake,
Izumida Yoshihiko,
Mitamura Keitaro,
Enami Yuta,
Niiya Takashi,
Murai Noriyuki,
Kato Hirohisa,
Shimizu Yoshinori,
Kou Keitatsu,
Furukawa Yoshinori,
Matsusita Michiaki,
Todo Satoru,
Shioda Seiji,
Kusano Mitsuo
Publication year - 2008
Publication title -
hepatology research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.123
H-Index - 75
eISSN - 1872-034X
pISSN - 1386-6346
DOI - 10.1111/j.1872-034x.2007.00311.x
Subject(s) - hepatocyte , cryopreservation , collagenase , transplantation , chemistry , intracellular , microbiology and biotechnology , biochemistry , biology , in vitro , surgery , medicine , embryo , enzyme
Aim: Hepatocyte transplantation is a potential alternative to whole organ liver transplantation. To realize this procedure, a hepatocyte bank system capable of supplying large numbers of hepatocytes must be established. We previously reported an easy method for cryopreserving hepatocytes using a microencapsulation technique. Here, we investigated how cryoinjury to microencapsulated hepatocytes could be avoided during cryopreservation. Methods: Hepatocytes from Sprague–Dawley rats were harvested in situ using a two‐step ethylenediaminetetraacetic acid (EDTA)/collagenase digestion protocol. The cells were microencapsulated using alginate‐poly L‐lysine. The microencapsulated hepatocytes were put into vials and immediately immersed in liquid nitrogen. The growth of ice crystals in the vials containing the microencapsulated hepatocytes was observed using cryomicroscopy. The microencapsulated hepatocytes were sectioned for ultrastructural examination to investigate their intracellular conditions. Finally, total RNA was isolated from the cryopreserved microencapsulated hepatocytes and analyzed for hepatocyte nuclear factor (HNF) using reverse transcriptase polymerase chain reaction (RT–PCR) analysis. Results: Cryomicroscopy showed that the alginate microencapsulation technique protected the hepatocytes from physical damage caused by the growth of extracellular ice crystals. Ultrastructural examination revealed that the intracellular environment of the microencapsulated hepatocytes was maintained. The RT–PCR analysis additionally suggested that the alginate gel also maintained the HNF level. Conclusion: Our microencapsulation technique protects hepatocytes from cryoinjury. This novel technique could be utilized by hepatocyte banks.