title: Cryopreservation of hepatocyte (HepG2) cell monolayers: impact of trehalose.
abstract: A simple method to cryogenically preserve hepatocyte monolayers is currently not available but such a technique would facilitate numerous applications in the field of biomedical engineering, cell line development, and drug screening. We investigated the effect of trehalose and dimethyl sulfoxide (Me2SO) in cryopreservation of human hepatocellular carcinoma (HepG2) cells in suspension and monolayer formats. HepG2 cell monolayers were incubated for 24h at varying concentrations of trehalose (50-150 mM) prior to cryopreservation to identify the optimum concentration for such preincubation. When trehalose alone was used as the cryoprotective agent (CPA), cells in monolayer format did not survive freezing while cells in suspension demonstrated 14% viability 24h after thawing. Only 6-13% of cells in monolayers survived freezing in cell culture medium supplemented with 10% Me2SO, but 42% of cells were recovered successfully if monolayers were preincubated with 100 mM trehalose prior to freezing in the Me2SO supplemented medium. Interestingly, for cells frozen in suspension in presence of 10% Me2SO, metabolic activity immediately following thawing did not change appreciably compared to unfrozen control cells. Finally, Raman spectroscopy techniques were employed to evaluate ice crystallization in the presence and absence of trehalose in freezing solutions without cells because crystallization may alter the extent of injury observed in cell monolayers. We speculate that biomimetic approaches of using protective sugars to preserve cells in monolayer format will facilitate the development of techniques for long-term preservation of human tissues and organs in the future.
authors.affiliation: Department of Biological Science, Eastern Illinois University, Charleston, Il 61920, United States.
authors.forename: Blake
authors.initials: B
authors.lastname: Stokich
authors.affiliation: Department of Mechanical Engineering, University of Michigan, Dearborn, MI 48128, United States.
authors.forename: Quinn
authors.initials: Q
authors.lastname: Osgood
authors.affiliation: Department of Biological Science, Eastern Illinois University, Charleston, Il 61920, United States.
authors.forename: David
authors.initials: D
authors.lastname: Grimm
authors.affiliation: Department of Biological Sciences, Division of Cellular, Developmental and Integrative Biology, Louisiana State University, Baton Rouge, LA 70803, United States.
authors.forename: Shhyam
authors.initials: S
authors.lastname: Moorthy
authors.affiliation: Department of Mechanical Engineering, University of Michigan, Dearborn, MI 48128, United States. Electronic address: nilay@umich.edu.
authors.forename: Nilay
authors.initials: N
authors.lastname: Chakraborty
authors.affiliation: Department of Biological Science, Eastern Illinois University, Charleston, Il 61920, United States. Electronic address: mmenze@eiu.edu.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Menze
mesh_terms: D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D005615:Freezing; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D014199:Trehalose / Q000378:metabolism*
chemical_list: D003451:Cryoprotective Agents; D014199:Trehalose; D004121:Dimethyl Sulfoxide
keywords: Adherent cell monolayer; DMSO; Osmotic stress; Respiration (OXPHOS)