Microencapsule technique protects hepatocytes from cryoinjury
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 mi...
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Published in | Hepatology research Vol. 38; no. 6; pp. 593 - 600 |
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Main Authors | , , , , , , , , , , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Melbourne, Australia
Blackwell Publishing Asia
01.06.2008
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Subjects | |
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Abstract | 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. |
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AbstractList | 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. 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. AIMHepatocyte 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. METHODSHepatocytes 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. RESULTSCryomicroscopy 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. CONCLUSIONOur microencapsulation technique protects hepatocytes from cryoinjury. This novel technique could be utilized by hepatocyte banks. 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. 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. 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. Our microencapsulation technique protects hepatocytes from cryoinjury. This novel technique could be utilized by hepatocyte banks. |
Author | Kusano, Tomokazu Enami, Yuta Yamada, Kousuke Niiya, Takashi Aoki, Takeshi Furukawa, Yoshinori Nishino, Nobukazu Shimizu, Yoshinori Koizumi, Tomotake Kusano, Mitsuo Shioda, Seiji Murai, Noriyuki Jin, Zhenghao Izumida, Yoshihiko Matsusita, Michiaki Odaira, Masanori Yasuda, Daisuke Hayashi, Ken Matsumoto, Shuichiro Kou, Keitatsu Todo, Satoru Mitamura, Keitaro Kato, Hirohisa |
Author_xml | – sequence: 1 givenname: Tomokazu surname: Kusano fullname: Kusano, Tomokazu email: kpochitomo@yahoo.co.jp organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 2 givenname: Takeshi surname: Aoki fullname: Aoki, Takeshi organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 3 givenname: Daisuke surname: Yasuda fullname: Yasuda, Daisuke organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 4 givenname: Shuichiro surname: Matsumoto fullname: Matsumoto, Shuichiro organization: First Department of Surgery, Hokkaido University School of Medicine, Hokkaido – sequence: 5 givenname: Zhenghao surname: Jin fullname: Jin, Zhenghao organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 6 givenname: Nobukazu surname: Nishino fullname: Nishino, Nobukazu organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 7 givenname: Ken surname: Hayashi fullname: Hayashi, Ken organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 8 givenname: Masanori surname: Odaira fullname: Odaira, Masanori organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 9 givenname: Kousuke surname: Yamada fullname: Yamada, Kousuke organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 10 givenname: Tomotake surname: Koizumi fullname: Koizumi, Tomotake organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 11 givenname: Yoshihiko surname: Izumida fullname: Izumida, Yoshihiko organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 12 givenname: Keitaro surname: Mitamura fullname: Mitamura, Keitaro organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 13 givenname: Yuta surname: Enami fullname: Enami, Yuta organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 14 givenname: Takashi surname: Niiya fullname: Niiya, Takashi organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 15 givenname: Noriyuki surname: Murai fullname: Murai, Noriyuki organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 16 givenname: Hirohisa surname: Kato fullname: Kato, Hirohisa organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 17 givenname: Yoshinori surname: Shimizu fullname: Shimizu, Yoshinori organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo – sequence: 18 givenname: Keitatsu surname: Kou fullname: Kou, Keitatsu organization: Electron Microscope Laboratory – sequence: 19 givenname: Yoshinori surname: Furukawa fullname: Furukawa, Yoshinori organization: Institute of Low-temperature Science, Hokkaido University, Hokkaido, Japan – sequence: 20 givenname: Michiaki surname: Matsusita fullname: Matsusita, Michiaki organization: First Department of Surgery, Hokkaido University School of Medicine, Hokkaido – sequence: 21 givenname: Satoru surname: Todo fullname: Todo, Satoru organization: First Department of Surgery, Hokkaido University School of Medicine, Hokkaido – sequence: 22 givenname: Seiji surname: Shioda fullname: Shioda, Seiji organization: First Department of Anatomy, Showa University School of Medicine, Tokyo and – sequence: 23 givenname: Mitsuo surname: Kusano fullname: Kusano, Mitsuo organization: General and Gastroenterological Surgery, Showa University School of Medicine, Tokyo |
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A potential treatment for liver disease publication-title: Dig Dis Sci – volume: 4 start-page: 303 year: 1968 end-page: 8 article-title: Mechanism of hemolysis of erythrocytes by freezing at near‐zero temperature publication-title: Cryobiology – volume: 12 start-page: 579 year: 2003 end-page: 92 article-title: Hypothermic storage and cryopreservation of hepatocytes publication-title: Cell Transplant – volume: 16 start-page: 105 year: 2000 end-page: 16 article-title: Viability and drug metabolism capacity of alginate‐entrapped hepatocytes after cryopreservation publication-title: Cell Biol Toxicol – volume: 12 start-page: 579 year: 2003 end-page: 92 article-title: Hypothermic storage and cryopreservation of hepatocytes: the protective effect of alginate gel against cell damage publication-title: Cell Transplant – volume: 11 start-page: 553 year: 2002 end-page: 61 article-title: Intrasplenic transplantation of encapsulated cells: a novel approach to cell therapy publication-title: Cell Transplantation – volume: 14 start-page: 609 year: 2005 end-page: 15 article-title: A novel method of cryopreservation of rat and human hepatocytes by using encapsulation technique and possible use for cell transplantation publication-title: Cell Transplant – volume: 10 start-page: 855 year: 1989 end-page: 60 article-title: Development and evaluation of a system of microencapsulation of primary rat hepatocytes publication-title: Hepatology – volume: 11 start-page: 28 year: 1953 end-page: 36 article-title: The mechanism of the protective action of glycerol against haemolysis by freezing and thawing publication-title: Biochem Biophys Acta – volume: 12 start-page: 607 year: 2003 end-page: 16 article-title: Cryopreservation of primarily isolated porcine hepatocytes with UW solution publication-title: Cell Transplant – volume: 4 start-page: 23 year: 2000 end-page: 40 article-title: Cryopreservation of marine algae publication-title: Recent Adv Mar Biotechnol – volume: 15 start-page: 826 year: 1987 end-page: 32 article-title: Cryopreservation of rat and dog hepatocytes for studies of xenobiotic metabolism and activation Drug Metab publication-title: Disposition – volume: 192 start-page: 892 year: 1976 end-page: 4 article-title: Hepatocellular transplantation for metabolic deficiencies: decrease of plasma bilirubin in Gunn rats publication-title: Science – ident: e_1_2_6_2_2 doi: 10.1177/096368979300200109 – ident: e_1_2_6_14_2 doi: 10.1016/0011-2240(90)90045-6 – ident: e_1_2_6_7_2 doi: 10.1126/science.818706 – ident: e_1_2_6_23_2 doi: 10.3727/000000002783985549 – ident: e_1_2_6_16_2 doi: 10.3727/000000005783982710 – ident: e_1_2_6_28_2 doi: 10.1016/S0011-2240(68)80128-2 – ident: e_1_2_6_12_2 doi: 10.1023/A:1007690009927 – ident: e_1_2_6_21_2 doi: 10.1016/j.jcrysgro.2004.10.085 – ident: e_1_2_6_8_2 doi: 10.1016/0011-2240(92)90048-7 – ident: e_1_2_6_18_2 doi: 10.1016/S0091-679X(08)61797-5 – ident: e_1_2_6_17_2 doi: 10.3727/000000007783464489 – ident: e_1_2_6_20_2 doi: 10.1002/hep.1840100518 – ident: e_1_2_6_4_2 doi: 10.1007/BF01307530 – ident: e_1_2_6_6_2 doi: 10.1016/j.jhep.2004.04.009 – volume: 338 start-page: 1422 year: 2004 ident: e_1_2_6_5_2 article-title: Treatment of Crigler–Najjar syndrome type I with hepatocyte transplantation publication-title: N Engl J Med doi: 10.1056/NEJM199805143382004 contributor: fullname: Fox IJ – ident: e_1_2_6_13_2 doi: 10.1002/bit.260430714 – volume: 4 start-page: 23 year: 2000 ident: e_1_2_6_26_2 article-title: Cryopreservation of marine algae publication-title: Recent Adv Mar Biotechnol contributor: fullname: Kazuka K – ident: e_1_2_6_25_2 doi: 10.1016/0006-3002(53)90005-5 – ident: e_1_2_6_27_2 doi: 10.1146/annurev.bb.03.060174.002013 – ident: e_1_2_6_19_2 doi: 10.3727/000000003108747181 – ident: e_1_2_6_10_2 doi: 10.1097/00007890-199303000-00028 – ident: e_1_2_6_15_2 doi: 10.3727/000000003108747217 – volume: 15 start-page: 826 year: 1987 ident: e_1_2_6_9_2 article-title: Cryopreservation of rat and dog hepatocytes for studies of xenobiotic metabolism and activation Drug Metab publication-title: Disposition contributor: fullname: Powis G – ident: e_1_2_6_3_2 doi: 10.1002/hep.510280527 – ident: e_1_2_6_24_2 doi: 10.1299/kikaib.67.580 – ident: e_1_2_6_11_2 doi: 10.3727/000000003108747181 – volume: 10 start-page: 267 year: 1996 ident: e_1_2_6_22_2 article-title: Liver‐enriched transcription factors and hepatocyte differentiation publication-title: FASEB J doi: 10.1096/fasebj.10.2.8641560 contributor: fullname: Silvia C |
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