The use of mouse embryonic stem (ES) cells in transgenic mouse production has contributed to a virtual explosion in the number of existing transgenic mouse models that are vital for human biomedical research [1; 2]. Coordinated projects to systematically knock out all mouse genes, such as the Knockout Mouse Project (KOMP) , Canada’s North American Conditional Mouse Mutagenesis Program (NorCOMM, http://norcomm.phenogenomics.ca/index.htm), the European Conditional Mouse Mutagenesis Program (EUCOMM, http://www.eucomm.org) , and Bay Genomics’ use of N-ethyl-N-nitrosurea (ENU) (http://baygenomics.ucsf.edu/) , will create thousands of mutant ES cell lines as a step towards producing mutant mice that serve as important models of human biology and disease . The C57BL/6 mouse lineage is central to these projects, due to its ease of genetic manipulation, wide accessibility to researchers, and the existence of ES cell lines of this genotype. Storage and maintenance of valuable genotypes as live animal lines would be wholly impractical . On the other hand, banking lines as ES cells is cost-effective and restoration of the ES cells into live, reproductively viable mice is routine in many laboratories across the world. Efficiency of this restoration is greatly improved when freezing and thawing methods produce healthy, rapidly dividing, germ line competent cells.
To our knowledge, there are no published reports quantifying post-thaw recovery of cryopreserved C57BL/6 ES cells. Post-thaw recovery of viable mouse ES cells varies dramatically from one cell line to the next, ranging anywhere from 10–90% (personal communication, Deanna Nielsen, Stem Cell Technologies technical support, 2004; personal communication, Xin Yu, University of California-Davis, 2004). Based on our experience, fewer than 50% of all cells in most mouse ES cell lines survive cryopreservation, which calls into question the efficacy of current methods. Cryopreservation protocols optimized to individual cell lines, if necessary, would allow for the full exploitation of ES cells from all strains, reducing the need for backcrossing in the production of mutant mice as individual backgrounds would be accessible as viable ES cell lines. Optimized cryopreservation protocols maximize post-thaw recovery of intact, pluripotent cells, reducing the time it takes to expand cultures post-thaw and increasing the number of aliquots one can cryopreserve from a single plate.
The cryobiological approach currently used to preserve ES cells is an equilibrium cooling approach. Equilibrium cooling relies on the formation of extracellular ice which leads to progressive cell dehydration, effectively concentrating the intracellular solution to a vitrifiable state in which, upon further cooling, the cytoplasm becomes a glass . The term, “vitrification” is most often used to describe cooling protocols which bring about an extreme elevation in viscosity of an extracellular solution, i.e. the formation of a glass in the absence of ice crystallization . Typically, this requires rapid cooling protocols in the presence of high concentrations of cryoprotective agents (CPAs). However, the use of equilibrium cooling to render a cell to a more vitrifiable state through controlled dehydration, an idea elucidated by Pegg and Diaper in 1990 , has only recently gained consistent and systematic support [10; 11; 12]. Major damage during a cryobiological protocol using the equilibrium cooling approach is theorized to be due to three major factors: osmotic damage due to water influx and efflux during the addition and removal of CPAs (e.g. Me2SO ), mechanical damage due to intracellular ice crystal formation, and solute effects, generally described as chemical damage that occurs due to an increase in the concentration of intracellular ions that occurs during freezing . The degree of damage caused by these factors varies with CPA and cooling and warming profiles.
A direct means to derive optimal cryopreservation protocols for a given cell type is via an exploration of the cell’s fundamental cryobiological parameters and how they relate to the major physical events that occur during the freezing process . Fundamental cryobiology, as it is applied to equilibrium cooling, seeks to define basic cell membrane permeability coefficients such as hydraulic conductivity (Lp), solute permeability (in our case, the permeability of cryoprotectants, PCPA) and their temperature dependence (defined by an Arrhenius equation with activation energy Ea) in order to predict optimal cooling and warming rates for that cell type. To avoid damage during the addition and removal of CPA, osmotic tolerance limits, nominal limits to which a cell can shrink or swell in response to osmotic stress without significant loss of function, are also defined. Each of these parameters can vary greatly according to cell type, species, and even individual (e.g. canine erythrocytes vs. spermatozoa of mice vs. chimpanzee spermatozoa [16; 17; 18]). Correspondingly, optimal cooling and warming rates can also vary greatly between these groups (e.g. human cord blood stem cells vs. erythrocytes [19; 20]).
Optimal cooling rates are defined as those that cool cell suspensions as rapidly as possible without causing a large difference between the intracellular freezing point and the intracellular temperature, and warming rates are classically optimal if they mimic their cooling rate counterparts . Using principles of the Boyle Van’t Hoff relation , the Arrhenius relation , a two-parameter mass transport model , and Mazur’s two factor hypothesis , we explored the fundamental cryobiological parameters of a C57BL/6 mouse ES cell line in order to improve existing cryopreservation protocols and define methods by which cryopreservation methods would be routinely assessed in a repository setting. PCPA and Lp in the presence of CPA (LpCPA), and the temperature dependence of these values were assessed in the presence of four commonly used CPA, namely ethylene glycol (EG), propylene glycol (PG), Me2SO, and glycerol (GLY). The osmotically inactive fraction of the cell, Vb, was calculated and osmotic tolerance limits were established using membrane integrity as the endpoint. These parameters were in turn used to predict a theoretically optimized cryopreservation protocol that improved the efficiency of cryopreservation for the C57BL/6 mouse ES cell line by greater than twofold. Finally, this protocol will provide an experimental basis to improve cryopreservation methods for other mouse ES cell lines, and will allow optimization accounting for other endpoints such as gene expression profiles associated with differentiation.