title: In vitro analysis of cryopreserved alginate-poly-L-lysine-alginate-microencapsulated human hepatocytes.
abstract: BACKGROUND
  The availability of well-characterized human hepatocytes that can be frozen and thawed will be critical for cell therapy. We addressed whether human hepatocytes can recover after microencapsulated cryopreservation and investigated whether these cryopreserved microencapsulated hepatocytes can be used for clinical applications.
  
  
  METHODS
  Adult hepatocytes of 18 separate donors were isolated with a two-step extracorporeal collagenase perfusion technique. After pre-incubation at 4 degrees C for 12-24 h in HepatoZYME-SFM, hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules. The microencapsulated hepatocytes were transferred to a complete medium containing 10% dimethyl sulphoxide. They were immediately placed into an isopropanol progressive freezing container at -80 degrees C overnight and immersed in liquid nitrogen the next day. During the post-thawing culture period, albumin secretion, urea synthesis, cell cycle, mRNA and protein levels, as well as the morphology and pathology structure of pre-incubation before microencapsulated cryopreservation (PMC) groups were analysed.
  
  
  RESULTS
  Compared with the immediate cryopreservation (IC) groups, we found significant improvement in the mRNA and protein levels in the attached cells, and higher secretion of albumin and urea levels after thawing. In the attached cultured human cryopreserved/thawed hepatocytes from the PMC group, albumin production was not significantly different from those of the direct culture groups on days 2, 3 and 4. The preserved morphology in the PMC group compared with the IC group was obvious.
  
  
  CONCLUSIONS
  The results of the present study suggested recovery of the functional and morphological integrity of human hepatocytes after pre-incubation at 4 degrees C for 12-24 h before microencapsulated cryopreservation. These studies offer the possibility for clinical applications in pharmacotoxicology, bioartificial liver and cell therapy in humans.
authors.affiliation: Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital, Medical College of Nanjing University, Jiangsu Province's Key Medical Center for Hepatobiliary Disease, Nanjing, China.
authors.forename: Hualian
authors.initials: H
authors.lastname: Hang
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Shi
authors.forename: Guang xiang
authors.initials: Gx
authors.lastname: Gu
authors.forename: Yafu
authors.initials: Y
authors.lastname: Wu
authors.forename: Jinyang
authors.initials: J
authors.lastname: Gu
authors.forename: Yitao
authors.initials: Y
authors.lastname: Ding
mesh_terms: D000328:Adult; D000464:Alginates / Q000494:pharmacology; D000704:Analysis of Variance; D015153:Blotting, Western; D002214:Capsules / Q000494:pharmacology; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000502:physiology; D002470:Cell Survival / Q000502:physiology*; D017690:Cell Transplantation / Q000379:methods; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D006498:Hepatectomy / Q000379:methods; D022781:Hepatocytes / Q000166:cytology* / Q000637:transplantation*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D011107:Polylysine / Q000031:analogs & derivatives / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014019:Tissue Donors
chemical_list: D000464:Alginates; D002214:Capsules; C051546:alginate-polylysine-alginate; D011107:Polylysine