Cryopreservation and quality control of mouse embryonic feeder cells q Ulf Diekmann a , Ralf Spindler b , Willem F. Wolkers b , Birgit Glasmacher b , Thomas Müller a,⇑ a Institute for Transfusion Medicine, Hannover Medical School, Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany b Institute of Multiphase Processes, Leibniz University Hannover, Callinstr. 36, D-30455 Hannover, Germany article info Article history: Received 28 October 2010 Accepted 8 July 2011 Available online 24 July 2011 Keywords: MEF Cryopreservation Quality control ESC abstract Stem cell research is a highly promising and rapidly progressing field inside regenerative medicine. Embryonic stem cells (ESCs), reprogrammed ‘‘induced pluripotent’’ cells (iPS), or lately protein induced pluripotent cells (piPS) share one inevitable factor: mouse embryonic feeder cells (MEFs), which are com- monly used for ESC long term culture procedures and colony regeneration. These MEFs originate from dif- ferent mouse strains, are inactivated by different methods and are differently cryopreserved. Incomprehensibly, there are to date no established quality control parameters for MEFs to insure consis- tency of ESC experiments and culture. Hence, in this work, we developed a bench-top quality control for embryonic feeder cells. According to our findings, MEFs should be inactivated by irradiation (30 Gy) and cryopreserved with optimal 10% DMSO at 1 K/min freezing velocity. Thawed cells should be free of mycoplasma and should have above 85 ± 13.1% viability. Values for the metabolic activity should be above 150 ± 10.5% and for the combined gene expression of selected marker genes above 225 ± 43.8% compared to non-irradiated, cryo- preserved controls. Cells matching these criteria can be utilized for at least 12 days for ESC culture with- out detaching from the culture dish or disruption of the cell layer. Ó 2011 Elsevier Inc. All rights reserved. Introduction Stem cell research holds great promise in regenerative medicine, especially after the discovery that pluripotency can be induced in certain somatic cell types using new reprogramming techniques [3,17,28]. However, results in differentiation and reprogramming success are highly variable depending e.g. on the somatic cell type, the age of the patient and the utilized protocol [30]. Mouse embryonic fibroblast feeder cells (MEFs) are often used as support- ive layer to culture embryonic stem cells (ESC), induced pluripotent cells (iPS), protein induced pluripotent cells (piPS) or fusion induced pluripotent cells [10,25,36,38] as well as a tool for ESC colony renewal after feeder-free cultivation. Feeder cells produce a unique cocktail of soluble molecules, like cytokines, of which the most important are Gremlin, Activin A and TGFb, that maintain the pluripotent state of ESCs [13]. Although several groups report culture methods for iPS or ESCs in feeder-free culture media [4,7,9,21] with similar gene-expression patterns compared to the classical feeder-based protocols [37], most of these techniques are hitherto not routinely used. Otherwise, feeder free procedures for differentiation of ESCs are well established [32]. The quality of feeder cells significantly influences long term growth and proliferation of undifferentiated ESCs by providing a complex mixture of cytokines and specific cell–cell interactions [13]. Therefore, two procedures greatly affect the quality of MEFs: (1) inhibition from proliferation by c-irradiation and (2) cryopres- ervation. Especially the inhibition from proliferation of MEFs is im- port to avoid overgrowth of the ESCs during the cultivation period. We have previously shown that c-irradiated MEFs are superior to Mitomycin inactivated MEFs for the cultivation of non-human pri- mate stem cells [13]. The exact mechanism of cellular damage caused by c-irradiation is speculative but is likely associated with the accumulation of free radicals reacting with double bonds of essential biomolecules which leads to an activation of the apopto- tic pathways. It is likely that irradiation may result in degradation of cellular biomolecules like lipids, proteins and DNA. Cryopreservation can lead to cellular damage as well. Cryoinju- ry can be divided into damage resulting from ‘‘solution effects’’ [12,24] and intracellular ice formation (IIF) [1,2,15]. Low cooling rates may lead to damage due to solution effects whereas IIF predominates at higher cooling rates. One of the paradigms in cryobiology is that there is an optimal cooling rate where damage due to solution effects and IIF are minimal yielding maximal sur- vival after thawing [14]. Cryopreservation strategies are based on minimizing cryoinjury by optimizing cooling and warming rates as well as the use of cryoprotective agents (CPAs). One special 0011-2240/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2011.07.002 q Statement of funding: This work is supported by funding from the German Research Foundation (DFG) for the Cluster of Excellence REBIRTH (From Regener- ative Biology to Reconstructive Therapy) and from the Institute for Transfusion Medicine of the Hannover Medical School. ⇑ Corresponding author. E-mail address: mueller.thomas@mh-hannover.de (T. Müller). Cryobiology 63 (2011) 104–110 Contents lists available at ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo