title: A novel method of cryopreservation of rat and human hepatocytes by using encapsulation technique and possible use for cell transplantation.
abstract: Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37 degrees C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.
authors.affiliation: Second Department of Surgery, School of Medicine, Showa University, Tokyo, Japan. takejp@wb4.so-net.ne.jp
authors.forename: Takeshi
authors.initials: T
authors.lastname: Aoki
authors.forename: Tomotake
authors.initials: T
authors.lastname: Koizumi
authors.forename: Yasuna
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yasuda
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Izumida
authors.forename: Zhenghao
authors.initials: Z
authors.lastname: Jin
authors.forename: Nobukazu
authors.initials: N
authors.lastname: Nishino
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Shimizu
authors.forename: Hirohisa
authors.initials: H
authors.lastname: Kato
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Murai
authors.forename: Takashi
authors.initials: T
authors.lastname: Niiya
authors.forename: Yuta
authors.initials: Y
authors.lastname: Enami
authors.forename: Keitaro
authors.initials: K
authors.lastname: Mitamura
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Kusano
mesh_terms: D000464:Alginates; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D002470:Cell Survival / Q000502:physiology; D017690:Cell Transplantation / Q000379:methods; D015925:Cryopreservation / Q000379:methods*; D051544:Cytochrome P-450 CYP3A; D022781:Hepatocytes / Q000166:cytology / Q000637:transplantation*; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D029362:Organic Anion Transporters, Sodium-Independent / Q000096:biosynthesis / Q000235:genetics; D011107:Polylysine / Q000031:analogs & derivatives; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen
chemical_list: D000464:Alginates; D008565:Membrane Proteins; D029362:Organic Anion Transporters, Sodium-Independent; D012333:RNA, Messenger; C088800:Slc22a7 protein, rat; C051546:alginate-polylysine-alginate; D011107:Polylysine; D001189:Aryl Hydrocarbon Hydroxylases; C465260:Cyp3a2 protein, rat; D051544:Cytochrome P-450 CYP3A