title: Long-term function of cryopreserved rat hepatocytes in a coculture system.
abstract: The goal of this study was to investigate postpreservation long-term function of cryopreserved primary rat hepatocytes using the hepatocyte/3T3-J2 fibroblast coculture system. The long-term function of thawed hepatocytes cocultured with fibroblasts was evaluated and compared with hepatocytes cultured without fibroblasts. Fresh isolated primary rat hepatocytes were frozen at a controlled rate (-1 degrees C/min) up to -80 degrees C, and then stored in liquid nitrogen for up to 90 days. Thawed hepatocytes were thereafter cocultured with 3T3-J2 murine fibroblasts and cocultivation was monitored for 14 days. The viability of fresh isolated hepatocytes was 91.4%, and that of cryopreserved hepatocytes was 82.1%. Cellular morphology and polarity, which were determined by the localization of actin filaments and connexin-32, were successfully maintained in cryopreserved hepatocytes following cryopreservation. Albumin and urea synthesis reached the maximum level and became stable after day 7 in coculture in both fresh and cryopreserved hepatocytes. Urea synthesis of cryopreserved hepatocytes was maintained 89.0% of nonfrozen fresh control, and albumin production of cryopreserved hepatocytes was 63.7% of control in coculture. Cytochrome P450 activity, which was measured by deethylation of ethoxyresorufin, was also maintained in cryopreserved hepatocytes at 88.6% of nonfrozen fresh control in coculture. The retention of synthetic and detoxification activities was verified to be well preserved during extended low-temperature storage (90 days). Both fresh control and cryopreserved hepatocytes cultured without fibroblast did not retain their synthetic and detoxification functions in long-term culture. These data illustrate that, through the utilization of our cryopreservation procedure, primary hepatocyte function was successfully maintained when placed into coculture configuration following thawing.
authors.affiliation: Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Keishi
authors.initials: K
authors.lastname: Sugimachi
authors.forename: Meindert N
authors.initials: MN
authors.lastname: Sosef
authors.forename: John M
authors.initials: JM
authors.lastname: Baust
authors.forename: Alex
authors.initials: A
authors.lastname: Fowler
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Tompkins
authors.forename: Mehmet
authors.initials: M
authors.lastname: Toner
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000418:Albumins / Q000096:biosynthesis; D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques; D003080:Cold Temperature; D017630:Connexins / Q000378:metabolism; D015925:Cryopreservation; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D005347:Fibroblasts; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D014508:Urea / Q000378:metabolism; D000097002:Gap Junction beta-1 Protein
chemical_list: D000418:Albumins; D017630:Connexins; D003577:Cytochrome P-450 Enzyme System; D014508:Urea; D000097002:Gap Junction beta-1 Protein