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Genetic Suppression of Cryoprotectant Toxicity T James R Cryobiology 86 (2019) 95–102 Contents lists available at ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/cryo Genetic suppression of cryoprotectant toxicity T James R. Cypsera, Wallace S. Chickb,c, Gregory M. Fahyd, Garrett J. Schumachera, ∗ Thomas E. Johnsona,e, a Institute for Behavioral Genetics, University of Colorado Boulder, USA b Department of Cell and Developmental Biology, University of Colorado Denver, Aurora, CO, USA c Charles C. Gates Center for Regenerative Medicine, University of Colorado Denver, Aurora, CO, USA d 21st Century Medicine, Inc., Fontana, CA, USA e Department of Integrative Physiology, University of Colorado Boulder, USA ARTICLE INFO ABSTRACT Keywords: We report here a new, unbiased forward genetic method that uses transposon-mediated mutagenesis to enable Cryoprotectant toxicity resistance the identification of mutations that confer cryoprotectant toxicity resistance (CTR). Our method is to select for Cryoprotective mutants resistance to the toxic effects of M22, a much-studied whole-organ vitrification solution. We report finding and Freezing injury characterizing six mutants that are resistant to M22. These mutants fall into six independent biochemical Transposons pathways not previously linked to cryoprotectant toxicity (CT). The genes associated with the mutations were Mutant selection Gm14005, Myh9, Nrg2, Pura, Fgd2, Pim1, Opa1, Hes1, Hsbp1, and Ywhag. The mechanisms of action of the Stem cells Mice mutations remain unknown, but two of the mutants involve MYC signaling, which was previously implicated in Forward genetics CT. Several of the mutants may up-regulate cellular stress defense pathways. Several of the M22-resistant mu- Stress resistance tants were also resistant to dimethyl sulfoxide (Me2SO), and many of the mutants showed significantly improved survival after freezing and thawing in 10% (v/v) Me2SO. This new approach to overcoming CT has many ad- vantages over alternative methods such as transcriptomic profiling. Our method directly identifies specific ge- netic loci that unequivocally affect CT. More generally, our results provide the first direct evidence that CT can be reduced in mammalian cells by specific molecular interventions. Thus, this approach introduces remarkable new opportunities for pharmacological blockade of CT. 1. Introduction [17,28], and the higher the concentration, the more likely it is that CT will arise. Further, since whole organs must be cooled relatively slowly The limited shelf life of donated organs negatively affects the suc- [12], this necessitates using relatively high cryoprotectant concentra- cess of human organ transplantation [1,22,35]. Efficient organ cryo- tions [28]. These high concentrations have high viscosity, which means preservation could potentially overcome many obstacles to organ slow introduction by perfusion and consequently relatively long ex- transplantation, thereby reducing costs, improving tissue matching, posure times to intrinsically toxic concentrations of cryoprotectant. reducing organ wastage, enabling elective scheduling of transplanta- Therefore, direct remedies for CT are needed for vitreous organ tion, and facilitating tolerance induction. It could also, in combination banking, and should have numerous practical advantages in cryo- with the generation of bioartificial organs or xenograft organ sources, biology in general. enable an end to the organ shortage. There is now good evidence that Here we describe an entirely new way of addressing the problem of the banking of large organs is possible CT. First, we created libraries of transposon-mutagenized mouse em- [3,5,14,21,24,29,31,42,54,57,60], but the toxicity of the cryoprotective bryonic stem cells (ESC). We then selected mutant cells that could agents needed to preserve certain organs, such as the kidney, appears to survive exposure to 9% of full-strength M22 (an organ vitrification be the major limiting factor [30]. solution; [21]) at 37 °C. This approach (illustrated in detail in Fig. 1) Cryoprotectant toxicity (CT) is particularly problematic during allows the cell itself to identify pathways that are crucial for reducing cryopreservation by vitrification (ice-free cryopreservation) [16,53], CT. We demonstrate here, for the first time, the existence of genes which has been gaining in popularity in recent years [23,27,28,35,45]. whose modulation can confer CT resistance (CTR). We also show that Vitrification requires extremely high concentrations of cryoprotectants the same genes that confer CTR also provide significant protection ∗ Corresponding author. Institute for Behavioral Genetics, University of Colorado Boulder, USA. E-mail address: [email protected] (T.E. Johnson). https://doi.org/10.1016/j.cryobiol.2018.11.003 Received 25 August 2018; Received in revised form 15 November 2018; Accepted 16 November 2018 Available online 17 November 2018 0011-2240/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). J.R. Cypser et al. Cryobiology 86 (2019) 95–102 product 15140122), beta-mercaptoethanol (55 μM, Thermo Fisher, product 21985-023) and 1000 unit per ml of leukemia inhibitory factor (AMSBio, product AMS-263-100). DPBS is Dulbecco's phosphate-buf- fered saline supplemented with calcium and magnesium (Gibco, pro- duct 14040-133). 2.2. Cells and cell culture conditions We generated a mouse ESC line from an F1 hybrid background (C57BL/6J × 129X1/SvJ), referred to here as “C9” [7,8]. For all ex- periments, cells were thawed and grown for 24 h under standard cul- ture conditions prior to harvesting for experimentation, unless other- wise stated. (We plated 100,000 cells in 5 ml ESC medium, and cultured cells at 37 °C [2].) 2.3. Toxicity of M22 to C9 cells at 2 °C Cells were trypsinized, divided into equal-volume aliquots of 150,000 cells, pelleted, and supernatants drawn off. The method of M22 addition and removal was modified from Guan et al [36]. All pellets were suspended in 333 μl ESC medium at 2 °C, and brought to 60% M22 by stepwise addition of 2 °C 100% M22 in LM5 to yield 10, 20, 40 and finally 60% M22, at 10-min intervals. After 0, 1, 2, 3, 4 and 8 h of Fig. 1. Overview of strategy for identifying and studying CTR mutants. Mutants exposure to 60% M22 at 2 °C, single samples were returned from 60% are generated and (1) selected for CTR. (2) Mutants are confirmed or rejected. M22 to near 0% M22 using sequential 10-min intervals. The M22 (3) Mutations putatively conferring CTR are identified. (4) Causality is verified concentration of each tube was adjusted to 30% by adding 600 mM by demonstrating that CTR is conferred by other mutations in the candidate mannitol solution (at 2 °C) in LM5 carrier solution, and then in sub- gene and is abolished by excising the pB transposon and (5) generating genomic sequent intervals, adjusted to 15, 7.5 and 3.75% M22, by adding equivalents of that mutation. (6) Bioinformatics are used to link the identified 300 mM mannitol (at 2 °C) in LM5 [36]. Following return to near 0% fi mutation to previously-identi ed pathways. (7) A search is made to identify M22, each sample was pelleted and cells were resuspended in 5 ml fresh known drugs that may induce CTR. Candidate drugs are then tested on wild- ESC medium and cultured in a T25 flask at 37 °C . At 48 h after the end type cells for efficacy in CTR. Drug effects are characterized (8) molecularly and of each exposure period, the numbers of cells surviving (adherent to the (9) phenotypically. (10) Ultimately, mice carrying the original mutation are floor of the flask) were counted. generated and novel improved drugs are developed using a variety of distinct methods, and (11) their organs are tested for CTR. 2.4. Mutagenesis and gene identification against freezing injury. Briefly, mouse ESC were mutagenized via random insertion of a piggyBac (pB) transposon [7,8]. We estimate that the mutant library 2. Materials and methods includes about 42,000 independent mutations (of which approximately 12,000 have been tested). Use of the pB transposon allows subsequent 2.1. Solutions excision from the genome (reversal of the mutation) to verify causality. Although most insertions cause loss of function, overexpression has also M22 is an 8-component vitrification solution developed by 21st been observed [8]. The mutant cell lines recovered remain hetero- Century Medicine (Fontana, CA) for the cryopreservation of kidneys zygous for the mutation unless treated further. Full details are available [21]. M22 was provided for these experiments by 21st Century Medi- elsewhere [8]. cine. M22 is used at concentrations approaching 9.4 M for the cryo- preservation of rabbit kidneys [24]. M22 is composed of 2.8 M Me2SO, 2.5. Selection for CTR 2.8 M formamide, 2.7 M ethylene glycol, 0.5 M N-methylformamide, 0.3 M 3-methoxy-1,2-propanediol, 2.8% (w/v) (less than 0.006 M) To select mutant cell lines resistant to CT, cells from the library were polyvinylpyrrolidone K12, 1% (w/v; less than 0.006 M) poly- plated in 15-cm plates at 6 × 106 cells per plate in 9% M22 and cul- vinylalcohol-polyvinylacetate copolymer [61], and 2% (w/v; less than tured at 37 °C for 48 h. Normal ESC medium without M22 was re- 0.03 M) polyglycerol [1]. We refer here to “% M22” (v/v) as the percent instated for 7 days of standard culture, after which six surviving co- of that maximum working concentration (9.4 M) used. lonies were picked and transferred to fresh individual flasks for LM5 is the carrier solution and diluent for M22, and was also pro- recovery and analysis. All pB insertion sites were determined by vided by 21st Century Medicine. It is an aqueous solution of 90 mM Splinkerette PCR [59]. This allowed identification of the genomic in- glucose, 45 mM mannitol, 45 mM lactose, 28.2 mM KCl, sertion sites associated with CTR.
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