Ming, last time we discussed mouse sperm cryopreservation. How is embryo cryopreservation different, and how should I choose between them?

 

Embryo cryopreservation involves superovulating female mice, mating them with target males, collecting 2-cell embryos, performing slow freezing, and storing them in -196°C liquid nitrogen. The choice depends on recovery considerations and cost factors. Let's explore this in detail.

 

Embryo Cryopreservation
 

Methods
 

Embryo cryopreservation is performed by superovulating female mice, mating them with target males, collecting 2-cell embryos, and subjecting them to slow freezing before storage in -196°C liquid nitrogen.
 

Advantages:  The method is reliable and well-established, preserves the complete genetic background of the strain, allows cryopreservation of homozygous embryos, and has a simple recovery protocol. Post-recovery embryo survival rates are high (>80%), with good pregnancy and birth rates after transplantation.
 

Disadvantages:  The procedure is technically challenging, requires specialized programmable freezing equipment, and is time-consuming.
Based on cooling rates, embryo cryopreservation methods include slow freezing, rapid freezing, and vitrification. Vitrification is the most commonly used method for mouse embryos.

 

Vitrification Principle
 

Embryos contain over 80% water, and ice crystal formation during freezing is the primary obstacle to successful cryopreservation.

Vitrification is an ultra-rapid freezing technique that uses high concentrations of cryoprotectants. During extremely rapid cooling, the cryoprotectant solution surrounding the embryo transforms into a high-viscosity, glass-like amorphous solid state.

In this vitrified state, the distribution of intracellular molecules and ions is preserved, avoiding the cellular damage caused by ice crystal formation in traditional slow freezing methods, thereby improving embryo survival rates.
 

Types of Cryoprotectants
 

The selection of cryoprotectants and control of cooling rates are critical factors in overcoming ice crystal formation during embryo cryopreservation.

Cryoprotectants are essential components in embryo cryopreservation technology, primarily categorized as:
 

Permeating Cryoprotectants:  Such as ethylene glycol (EG) and dimethyl sulfoxide (DMSO). These can enter cells and replace water, preventing intracellular ice crystal formation, but they have some toxicity to cells.
 

Non-Permeating Cryoprotectants:  Such as sucrose and Ficoll. These cannot freely cross cell membranes but increase extracellular osmotic pressure, promoting cellular dehydration and reducing osmotic shock during thawing.
 

Bioactive Substances:  Such as bovine serum albumin. These stabilize cell membrane structures, prevent zona pellucida hardening during freezing, and reduce the impact of osmotic pressure changes on embryos.
 

Vitrification Procedure
 

1.Filter 1M DMSO solution while placing four 100μL droplets of 1M DMSO in a culture dish. One droplet is for washing embryos from the collection medium, while the others are for placing washed embryos.

2.Transfer a group of embryos from the collection medium into one droplet, then distribute the washed embryos evenly among the other three droplets. These embryos will eventually be transferred to cryotubes.

3.Using a 20μL pipette with a gel tip, adjust to 5μL and aspirate all embryos. Transfer them into a cryotube, then place the cryotube in a desktop cooler at 0°C for 5 minutes.
 

Note:
 

●Cryotubes can remain in the 0°C cooler for up to 20 minutes if needed.

●Before aspirating embryos, gently swirl the culture dish to concentrate embryos in the center of the droplet, making it easier to aspirate all embryos in 5μL of 1M DMSO solution.

1.Add 45μL of pre-equilibrated antifreeze solution (DAP213) from the 0°C cooler, then equilibrate for another 5 minutes.

2.Quickly secure the cryotube in a fixation clamp and immediately immerse it in liquid nitrogen.

 

So how do I decide between embryo cryopreservation and sperm cryopreservation?

 

Choosing Between Embryo and Sperm Cryopreservation
 

1. Recovery Considerations
 

If you choose embryo cryopreservation, the recovered offspring will have the exact genetic background of the original frozen mouse strain. If you choose sperm cryopreservation, you must consider the egg donor during recovery. For example, if using B6 females, the recovered offspring will be at least heterozygous for one generation.
 

2. Cost Considerations
 

Embryo Cryopreservation:  The freezing process is time-consuming and expensive, but recovery costs are low.

Sperm Cryopreservation:  The freezing process is low-cost, fast, and time-efficient, but recovery via in vitro fertilization (IVF) is relatively expensive. However, sperm recovery can yield many offspring in a single session.
 

Which Preservation Method is Recommended?
 

1.Multi-gene homozygous mice:  Recommended for embryo cryopreservation. These mice require long-term breeding and screening processes. Embryo cryopreservation allows using homozygous males and females of the same strain to obtain embryos, and recovery yields offspring with the original genotype.

2.Single-gene mice and multi-gene heterozygous mice:  Recommended for sperm cryopreservation. Recovery can use wild-type females or genetically engineered females as required for the experiment.

3.Case-by-case consideration:  The specific choice often depends on the available animal numbers and specific experimental requirements.
 

References:
 

[1] Tsinghua University  - Laboratory Animal Research Center

[2] ShanghaiTech University  - Laboratory Animal Center

[3] Nakao K., Nakagata N., and Katsuki M. 1997. Simple and efficient procedure for cryopreservation of mouse embryos by simple vitrification. Exp. Anim. 46: 231-234.

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