Archiving genetically altered animals: a review of cryopreservation and recovery methods for genome edited animals

Abstract

With the ever-expanding numbers of genetically altered (GA) animals created in this new age of CRISPR/Cas, tools for helping the management of this vast and valuable resource are essential. Cryopreservation of embryos and germplasm of GA animals has been a widely used tool for many years now, allowing for the archiving, distribution and colony management of stock. However, each year brings an array of advances, improving survival rates of embryos, success rates of in-vitro fertilisation and the ability to better share lines and refine the methods to preserve them. This article will focus on the mouse field, referencing the latest developments and assessing their efficacy and ease of implementation, with a brief note on other common genetically altered species (rat, zebrafish, Xenopus, avian species and non-human Primates).

With the ever-expanding numbers of genetically altered (GA) animals created in this new age, tools for helping the management of this vast and valuable resource are essential. Cryopreservation is a key tool to allow the preservation of valuable resources from breeding stagnation and loss, slowing genetic drift when used properly. It can allow the sharing of colonies with collaborators in an easy, timely, ethical and more cost effective manner and it can protect strains from loss of phenotype, whilst also saving money in the long run, reducing the use of animals. In mice, where the technology is most advanced, if a strain is frozen (where there is access to an in-vitro fertilisation (IVF) and embryo transfer service) it can be effectively managed from the freezer. This removes the need for ‘tick-over’ colonies and can assist with cleaning up a colony’s health status, making it a service that every large rodent facility should invest in. For those without access to such a service, there are cryobanking repositories^1,2 offering free archiving service as long as the line is made available for open access as well as commercial services. ^{3 –5}

A brief history of mouse cryopreservation

Cryopreservation of embryos and gametes has been in use in the preservation of GA animals for decades. The Jackson Laboratory set up the first successful mouse repository in 1976^{6, 7} on the back of the 1971 and 1972 publications by both Whittingham, Leibo and Wilmut on the successful freezing and thawing of mouse embryos using dimethyl sulfoxide (DMSO), producing live pups. ^{8 –11} In 1984, a publication described a method to obtain high embryo survival rates using 1–2 propanediol and sucrose combined with a slow rate freezing method. In 1990, Nakagata published a rapid freezing and thawing method using DAP213 cryoprotectant (2 M DMSO, 1 M acetamide, 3 M propylene glycol) in PB1,^12,13 building on work done by Rall and Fahy in 1985–1987. They published the first vitrification protocols, working to increase viability through the elimination of extracellular ice.^14,15 Kasai also developed this further to produce protocols based on a two-step process to maximise cryoprotectant permeation, using ethylene glycol, Ficoll and sucrose (EFS). ^{16 –18} These techniques are still some of the most widely used methods to freeze mouse embryos.

Mammalian sperm cryopreservation was largely successful from the 1950s and 60s, after the accidental discovery of the cryoprotective properties of glycerol by Polge, Smith and Parkes in 1949 (at the former MRC’s NIMR). ¹⁹ However, there was no real progress in the rodent field until 1990 when papers by Okuyama, Yokoyama and Tada ^{20 –23} all showed some success with a mix of cryoprotective agents. Over several publications, Nakagata modified the Okuyama technique and proved that whilst glycerol was ineffective at protecting mouse spermatozoa, a skimmed milk/raffinose cryoprotectant increased the fertilisation potential of most hybrid and outbred mouse lines.^22,24 In 1997, Sztein published a vital study that combined this method with a harvest temperature of 37 degrees to form the basis of what was then widely referred to as the Jax method for sperm cryopreservation. ²⁵ Whilst this method was widely used for many years, the increased use of inbred C57BL/6 sub-strains of mice to produce GA lines proved to be problematic, with greatly reduced fertilisation rates being seen on this background. Despite methods such as zona drilling improving fertilisation rates, further advances were needed.

In 2008 Ostermeier increased the fertility of frozen C57BL/6 sperm with the addition of monothioglycerol (MTG) and increased sperm capacitation time before IVF insemination. ²⁶ In 2011, the CARD group led by Nakagata published a new method that greatly increased the fertilisation potential of frozen C57BL/6 sub-strains of sperm. They added glutamine to the cryoprotectant (which has a protective effect maintaining post-thaw motility and reducing plasma membrane damage) and included methyl-beta-cyclodextrin (MBCD) to the sperm capacitation media to promote cholesterol efflux. Additionally, reduced-glutathione was added to the IVF media to alleviate oxidative stress, increasing fertilisation rates. ^{27 –31} A full IVF method was published in 2014. ³² This method is now widely used to cryopreserve GA mouse lines via sperm freezing with reliable and reproducible results across all backgrounds. Sztein, Takeo and Nakagata published a detailed history of the cryobiology of sperm in 2018. ²²

Established mouse techniques

Before we expand on the latest advances in the field, a quick recap. Embryo cryopreservation involves producing large numbers of embryos, often by superovulating (preferably young) females, mating them and then culling at day 2.5 to freeze flushed embryos at the robust 8-cell stage. Embryos are also frozen using IVF techniques whereby animals are superovulated, oocytes harvested and combined with sperm in the dish and fertilised 2-cell embryos are frozen. Controlled rate freezers, which allow slow freezing of embryos whilst in a cryoprotective medium, thereby avoiding ice damage, is a common and consistent mechanism. Other available methods include vitrification and rapid freezing methods. Vitrification has greater utility, is faster and can be inexpensive, requiring less specialised equipment and can avoid any damage from ice crystal formation if performed correctly. ^{33 –35} However, it is very operator dependent, and the high concentrations required to rapidly dehydrate the cells can be very damaging if the timing is not accurate. Thawing of vitrified cells is also a precise business, and the small volumes involved can thaw quickly if not properly stored.

Sperm cryopreservation uses far fewer animals to preserve a strain, as only two to four males are typically required, generally in the optimum age range of 8–24 weeks. Males are killed, vas deferens removed and sperm is released into a cryoprotective solution before being placed in containers and frozen in LN₂ (liquid nitrogen) vapour.

Advances in embryo cryopreservation and IVF

Embryo cryopreservation has remained much the same over the years, but the methods used to produce the embryos have been increasingly developed. Traditional methods involved superovulating and mating GA stock to produce embryos. Embryos can be frozen at any pre-implantation stage but are at their most robust at the 8-cell stage. However, since the advent of advanced mediums (such as human tubal fluid based medium (HTF) supplemented with reduced-glutathione (GSH)) to improve fertilisation rates, using IVF as the primary method for embryo production is becoming more mainstream. The advantages of needing fewer stud males and less reliance on mating success often outweighs the disadvantage of freezing at the more delicate 2-cell stage. ³⁵ There are several methods that can be used to boost fertilisation rates, including caffeine supplementation, ³⁶ laser-assisted zona drilling ³⁷ and utilising micro-droplets within the IVF³⁸ but a widely used rescue method is the CARD method, published in 2013 as a means to bring back lines frozen with suboptimal historical sperm freezing methods. ³⁹

One of the most innovative new techniques in recent years is the introduction of hyper-ovulation in 2015. ⁴⁰ Inhibin is the hormone that acts in the pituitary gland of mammals to prevent the secretion of follicle stimulating hormone (FSH), the hormone responsible for oocyte development in the ovary. ⁴¹ Hyper-ovulation involves co-injecting inhibin antiserum (iAS) with pregnant mare serum gonadotropin (PMSG) to prevent the secretion of inhibin, breaking this negative feedback loop. The signal to stop producing FSH becomes non-existent, and therefore the ovary continues to produce oocytes. Whilst superovulation already increases oocyte yield (a naturally mated female produces five to eight oocytes and superovulation increases this yield to 15–25 oocytes in C57BL/6J mice), by co-injecting iAS, this yield can be increased to 50–100 oocytes per female. Although it is relatively new, its use is already growing because of its capacity to cause a significant reduction in egg donor females, making it possible to rapidly archive GA lines via IVF using as little as two or three females. The optimal age for hyper-ovulation of female mice is 4 weeks.^42,43 Whilst this new technology is promising, there are still limitations to its use. Due to the dense cumulus-oocyte-complexes (COCs) produced when using iAS, its use is limited to IVF rather than in combination with mating, as the sperm is unable to penetrate the COCs in vivo. There is also an increased risk of ovarian hyper-stimulation syndrome (OHSS) in the mice and caution should be taken as with all new technologies to closely monitor the welfare of the animals, and batch test the iAS. More recent papers, however, show its effectiveness across a range of strains, and its potential for rescuing difficult models should not be overlooked.^44,45

Vitrification has been used successfully for over three decades, and its popularity is spreading as techniques have been improved and its benefits become more widely applicable in line with other advances. In 2013 Nakagata et al. published a method to freeze oocytes successfully using a pre-freeze incubation of 2% foetal calf serum in HTF. ⁴⁶ This technique allows creation of an archive of wild-type oocytes, allowing greater flexibility in IVF scheduling. The method has been developed further allowing reliable cryopreservation of zygotes. ^{47 –49} These advances in cryopreservation and IVF technology have widened the potential applications for its use from just archiving GA lines to supporting their production too. Depending on the mutation, GA production can require hundreds of zygotes to produce founders. Hyper-ovulation would greatly reduce the number of oocyte donors needed, and combining this with the ability to use vitrified zygotes provides a great deal of flexibility for this workflow. All this whilst reducing the number of animals used.

Whilst these developments are exciting in their potential, consistency in the application of these methods is still a barrier to their uptake and large-scale use.

Advances in sperm cryopreservation and associated technologies

The method of sperm cryopreservation has not changed in recent years, but there have been developments in the transportation of both mouse sperm and embryos to make it easier to distribute GA strains around the world. In 2012, Takeo described the efficiency of transporting the cauda epididymis of GA mice at chilled temperatures using Lifor preservation medium. This maintained good fertilisation rates for up to 96 hours, allowing transportation of sperm around the globe without the need to cryopreserve the line first.^50,51 Subsequent publications from Takeo proved that sperm transported in Lifor can be used both fresh in an IVF and also successfully frozen after transportation. ⁵² Additionally, sharing of archived material and germplasm has become much more accessible since the advances in shipping sperm on dry ice ⁵³ as well as the ability to share live embryos and sperm.^50,54 In 2018, a method of storing frozen sperm samples at –80°C for up to two years was described, alongside a method to freeze sperm at –80°C without the use of LN₂. These developments enable sperm cryopreservation to be used as a means to archive GA strains when resources, such as LN₂ storage capability, are not available or are limited.^55,56

Standard practice for sperm collection for both cryopreservation and IVF is to euthanize male mice and dissect the cauda epididymis. However, there are times when male mice of the correct genotype are scarce within a colony, making a terminal procedure an unattractive option. One option is to surgically remove a single cauda for the purpose of securing the line or using in an IVF for a more rapid expansion. This allows males on the shelf to continue breeding if necessary. ⁵¹ This procedure can only be performed once per male. An alternative is to perform serial sperm sampling via puncture of the epididymis, which can be performed multiple times for use in IVF without affecting the breeding potential of the males. ⁵⁸ In both cases, these invasive techniques would require a strong scientific justification given the ethical implications of the procedures.

A lesser-used technique is that of ovary cryopreservation. Whilst not often used (cryopreservation can damage the ovary reducing fertility; recovery is technically difficult and requires a histocompatible recipient) it is useful in strains where breeding females have difficulty or become ill at an early age, and to preserve oocytes, particularly when fertile males are in short supply. Ovaries are cryopreserved preferably from young females in halves, and once the technician is competent have a very good recovery rate. The ovarian transplant technique (without cryopreservation) can also be useful for increasing the number of breeding age females to rescue a strain. ⁶⁰

Records

It should be noted that there are some basic principles to be followed whatever species or methods are used to preserve a colony, especially when sharing stocks. It is essential that accurate and comprehensive notes are kept on all stocks being frozen (such as allele details, background, phenotype) as the scientific value of the stock depends on this. Many guidelines exist for the provision of an accurate GA passport, such as the Royal Society for the Prevention of Cruelty to Animals’ (RSPCA) ‘key to consistent care’ ⁶¹ encouraging sharing of husbandry, breeding and phenotyping as well as the use of accurate nomenclature. ^{62 –65} It is also essential to split stocks of archived material and look to apply quality control before a line is fully removed. ⁶⁵ Measures must be taken to check the health status of the imported stock, ⁵⁷ as well as the genetic background. ⁶⁵ If detailed information on the background sub-strain and breeding records are not available, it would be prudent to check via genotyping for the allele of interest as well as other commonly used alleles (e.g. Cre, reporters) which are often crossed into a stock and then forgotten. The use of Single nucleotide polymorphism (SNP) or Short tandem repeat (STR) panels to characterise the genetic background allows decisions to be made to ensure reproducibility of experiments. All the major commercial suppliers of mice and of genotyping services have an array of appropriate panels. Information can also be obtained on many strains from the relevant repositories, resource centre and phenotyping platforms.^5,66 There are also local mechanisms to source mice, such as the mouse locator scheme which is available in several countries. ⁶⁷

A note on other species

Zebrafish

Zebrafish (Danio rerio) have seen increasing popularity over the past 40 years and are now a staple model organism in biomedical research. This is due in part to their small size, high fecundity, rapid development and transparent external embryo.^68,69 Globally there are now three major zebrafish resource centres^62,70,71 who aim to act as centralised repositories for mutant and wild-type strains and standardise protocols across research facilities. As in murine research, the advent of reliable and efficient genome editing tools such as CRISPR/Cas has seen a boom in the number of complex GA lines being produced.^72,73 Maintaining these lines only in live breeding stocks is resource heavy and risky as it leaves colonies vulnerable to accidental loss as a result of pathogens, genetic drift or contamination. ⁷⁴

Currently there is no viable methodology for the cryopreservation of fish oocytes or embryos due to their large size and structural complexity.^75,76 Traditionally there has been a large variation in spermatozoa cryopreservation procedures between research labs, resulting in inconsistent thaw rates and the loss of lines. In the past decade significant efforts have enabled standardisation of protocols in larger institutes.^74,77–80 ZIRC (Zebrafish International Resource Center), the most well-established repository based in North America, ⁶² regularly publishes updates on the high-throughput methodology they have established, ⁶⁹ while the European (EZRC (European Zebrafish Resource Center)) and UK institutes use adaptions based on the protocol developed by the Wellcome Trust’s Sanger Institute.^80,81 There are also several successful methods available for medaka. ⁸²

Xenopus

Xenopus species have also been successfully cryopreserved and recovered ⁸³ although this has generally been less reliable in X. laevis than X. tropicalis.^84,85 The European Xenopus Resource Centre (EXRC) at the University of Portsmouth in the UK⁸⁷ and the National Xenopus Resource (NXR) at the Marine Biological Laboratory in Woods Hole, USA⁸⁸ work together to provide an archiving repository for the Xenopus community. ³⁴

Rats

Rats are less used than mice in research, but there is still a substantial and growing bank of models needing preservation. Embryo freezing is reliable and similar to mouse methods, but methods for rat sperm result in inconsistent recovery rates, largely due to the longer size of rat sperm compared to that of other species and that it is easily damaged during the freezing process.^88,89 Nakagata and Takeo, who have so transformed the mouse field, have turned their hand to rats in recent years achieving fertilisation rates of 80% with frozen sperm compared to 94% with fresh IVF. ⁹⁰ Using IVF to generate GA rats via electroporation is also increasingly successful, ⁹¹ and methods are also available from the rat repositories.^92,93

Avians

Germplasm cryopreservation can be challenging in avian species, where diversity is still best maintained through live colonies. As embryo cryopreservation is not an option for egg laying species, the focus swings to sperm, or precursor cells, embryonic stem cells (ESCs) and primordial germ cells (PGCs). Sperm can be very effective for preservation of a mutated allele, as well as preserving the genetic diversity of a species/strain. It is non-invasive, easy to preserve and reintroduce. However, it does not allow for the recovery of the full genetic background and success is very breed dependent. In chickens there has been some success in recent years in the preservation of PGCs^94,95 and together with sperm and reproductive tissues, national programmes of biobanking for avians are being developed at an increasing rate. ⁹⁶

Non-human Primates (NHPs)

When it comes to Primates, again there isn’t a commonly used technique, but there are a variety of methods available for collection and cryopreservation of NHP sperm. ⁹⁷ Artificial insemination (AI) seems to be moderately successful in certain species whereas IVF is variable and not universally applicable. ⁹⁸

Whatever method is used, and whether facilities set up their own specialist archiving service or utilise the many repositories set up and expanding across the world, cryopreservation is a key method that will continue to be utilised to help manage resources and, as such, the technology will continue to be developed to improve its efficacy and reliability.