Microencapsule technique protects hepatocytes from cryoinjury.
OBJECTIVE
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.


CONCLUSIONS
Our microencapsulation technique protects hepatocytes from cryoinjury. This novel technique could be utilized by hepatocyte banks.
Kusano
Tomokazu
T
General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo, Japan.
Aoki
Takeshi
T
Yasuda
Daisuke
D
Matsumoto
Shuichiro
S
Jin
Zhenghao
Z
Nishino
Nobukazu
N
Hayashi
Ken
K
Odaira
Masanori
M
Yamada
Kousuke
K
Koizumi
Tomotake
T
Izumida
Yoshihiko
Y
Mitamura
Keitaro
K
Enami
Yuta
Y
Niiya
Takashi
T
Murai
Noriyuki
N
Kato
Hirohisa
H
Shimizu
Yoshinori
Y
Kou
Keitatsu
K
Furukawa
Yoshinori
Y
Matsusita
Michiaki
M
Todo
Satoru
S
Shioda
Seiji
S
Kusano
Mitsuo
M