title: Systematic parameter optimization of a Me(2)SO- and serum-free cryopreservation protocol for human mesenchymal stem cells.
abstract: Human mesenchymal stem cells (hMSCs) have great potential for clinical therapy and regenerative medicine. One major challenge concerning their application is the development of an efficient cryopreservation protocol since current methods result in a poor viability and high differentiation rates. A high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of cryoprotective agents (CPA) in sufficient concentrations. The most widely used CPA, dimethylsulfoxide (Me(2)SO), is toxic at high concentrations at temperatures >4°C and has harmful effects on the biological functionality of stem cell as well as on treated patients. Thus, this study investigates different combinations of non-cytotoxic biocompatible substances, such as ectoin and proline, as potential CPAs in a systematic parametric optimization study in comparison to Me(2)SO as control and a commercial freezing medium (Biofreeze®, Biochrom). Using a freezing medium containing a low proline (1%, w/v) and higher ectoin (10%, w/v) amount revealed promising results although the highest survival rate was achieved with the Biofreeze® medium. Cryomicroscopic experiments of hMSCs revealed nucleation temperatures ranging from -16 to -25°C. The CPAs, beside Me(2)SO, did not affect the nucleation temperature. In most cases, cryomicroscopy revealed intracellular ice formation (IIF) during the cryopreservation cycle for all cryoprotocols. The occurence of IIF during thawing increased with the cooling rate. In case of hMSC there was no correlation between the rate of IIF and the post-thaw cell survival. After thawing adipogenic differentiation of the stem cells demonstrated cell functionality.
authors.affiliation: University of Applied Sciences Giessen - Friedberg, Institute of Bioprocess Engineering and Pharmaceutical Technology, Wiesenstrasse 14, 35390 Giessen, Germany. denise.freimark@tg.fh-giessen.de
authors.forename: Denise
authors.initials: D
authors.lastname: Freimark
authors.forename: Constanze
authors.initials: C
authors.lastname: Sehl
authors.forename: Christian
authors.initials: C
authors.lastname: Weber
authors.forename: Klaus
authors.initials: K
authors.lastname: Hudel
authors.forename: Peter
authors.initials: P
authors.lastname: Czermak
authors.forename: Nicola
authors.initials: N
authors.lastname: Hofmann
authors.forename: Ralf
authors.initials: R
authors.lastname: Spindler
authors.forename: Birgit
authors.initials: B
authors.lastname: Glasmacher
mesh_terms: D050156:Adipogenesis; D000599:Amino Acids, Diamino / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D002470:Cell Survival; D020285:Cryoelectron Microscopy; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000633:toxicity*; D005615:Freezing; D006801:Humans; D007053:Ice; D059630:Mesenchymal Stem Cells / Q000187:drug effects*; D011392:Proline / Q000494:pharmacology*
chemical_list: D000599:Amino Acids, Diamino; D003451:Cryoprotective Agents; D007053:Ice; C045628:ectoine; D011392:Proline; D004121:Dimethyl Sulfoxide