title: Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch-based cryoprotectants.
abstract: BACKGROUND
  Preservation of human skin fibroblasts and keratinocytes is essential for the creation of skin tissue banks. For successful cryopreservation of cells, selection of an appropriate cryoprotectant agent (CPA) is imperative. The aim of this study was to identify CPAs that minimize toxic effects and allow for the preservation of human fibroblasts and keratinocytes in suspension and in monolayers.
  
  
  RESULTS
  We cryopreserved human fibroblasts and keratinocytes with different CPAs and compared them to fresh, unfrozen cells. Cells were frozen in the presence and absence of hydroxyethyl starch (HES) or dimethyl sulfoxide (DMSO), the latter of which is a commonly used CPA known to exert toxic effects on cells. Cell numbers were counted immediately post-thaw as well as three days after thawing. Cellular structures were analyzed and counted by labeling nuclei, mitochondria, and actin filaments. We found that successful cryopreservation of suspended or adherent keratinocytes can be accomplished with a 10% HES or a 5% HES, 5% DMSO solution. Cell viability of fibroblasts cryopreserved in suspension was maintained with 10% HES or 5% HES, 5% DMSO solutions. Adherent, cryopreserved fibroblasts were successfully maintained with a 5% HES, 5% DMSO solution.
  
  
  CONCLUSIONS
  We conclude that skin tissue cells can be effectively cryopreserved by substituting all or a portion of DMSO with HES. Given that DMSO is the most commonly used CPA and is believed to be more toxic than HES, these findings are of clinical significance for tissue-based replacement therapies. Therapies that require the use of keratinocyte and fibroblast cells, such as those aimed at treating skin wounds or skin burns, may be optimized by substituting a portion or all of DMSO with HES during cryopreservation protocols.
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.|Interdisciplinary Institute for Bioinformatics, University of Leipzig, Leipzig, Germany.
authors.forename: Yahaira
authors.initials: Y
authors.lastname: Naaldijk
authors.affiliation: Department of Ophthalmology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Adiv A
authors.initials: AA
authors.lastname: Johnson
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.
authors.forename: Annett
authors.initials: A
authors.lastname: Friedrich-Stöckigt
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany. A.Stolzing@lboro.ac.uk.|Interdisciplinary Institute for Bioinformatics, University of Leipzig, Leipzig, Germany. A.Stolzing@lboro.ac.uk.|Centre for Biological Engineering, Wolfson School of Material and Manufacturing Engineering, Loughborough University, Loughborough, UK. A.Stolzing@lboro.ac.uk.
authors.forename: Alexandra
authors.identifier: http://orcid.org/0000-0002-2975-5736
authors.initials: A
authors.lastname: Stolzing
mesh_terms: D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000737:chemistry / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D006895:Hydroxyethyl Starch Derivatives / Q000494:pharmacology*; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D008297:Male; D012867:Skin / Q000166:cytology / Q000187:drug effects; D012879:Skin Physiological Phenomena / Q000187:drug effects
chemical_list: D003451:Cryoprotective Agents; D006895:Hydroxyethyl Starch Derivatives
keywords: Cryopreservation; Cryoprotectants; Dimethyl sulfoxide; Fibroblasts; Hydroxyethyl starch; Keratinocytes