Author: Lluís Montoliu
Company: CNB-CSIC

Lluís Montoliu (1995)

4.- CRYOPRESERVATION OF MOUSE EMBRYOS

	After generating a couple of transgenic lines and expanding them by breeding one realizes about the problem of space in the animal house. Moreover, once those lines have been published and no more work is planned for the immediate future with them there are basically two alternatives. Either keep them mating (with high costs in space, time and analysis to monitor every litter) or, eventually, discard them. What follows is a third and more interesting alternative. Mammalian embryos can be frozen away and stored indefinitely in liquid nitrogen. There are many protocols available. Some of them require expensive and complicate machines which will drop the temperature step-by-step, carefully. This protocol is easy, fast and do not requires any special equipment. It was originally designed for cow embryos and had to fulfil the criteria of being movable, and easy-to-perform, since most of the manipulations would be done in the farm (Walter Horlacher, 1993, PhD work). The name of the process is VITRIFICATION and was originally introduced in 1987 by Rall. The use of glycerol and other compounds impairs the formation of water crystals by almost complete dehydration. Recovery of the embryos is done by step-by-step re-hydration, exchanging back the glycerol by the original water.

	Procedure:					Reference: Cryobiology (1987) 24: 387-402

FREEZING

	The best embryos to cryopreserve by vitrification are compacted morulas. However, 8-cell embryos and early morulas can also be used with less efficiency. Compacted morulas (easy to discover since represent the transition between a characteristic 8-cell embryo and a ball of cells which have a tendency to diffuse the limits between them, i.e. like a raspberry) are found in the oviduct and, occasionally, in the uterus of pregnant mouse females. For best recoveries embryos should be collected 2 days after vaginal plug has been detected in the female. On the due day, it is better to open the female early in the afternoon (around 14:00 in an animal house based on 16:00-04:00 dark cycle). Embryos will be collected in M2 medium (see section 2.a) by flushing both horns of the uterus and disrupting the oviduct, as described in section 2.b. Finally, we should obtain a number of embryos (from the appropriate embryonic stage) in a clean drop of M2. Use the usual Pasteur pipettes connected by tubing to a mouth piece to move the embryos from drop to drop. Alternatively, use hand pipettes (fixed volume=5 μl) called Transferpettor (from Brand). Follow all manipulations under a binocular microscope set to medium magnification.
	Before starting the freezing steps with the embryos the capillar which will receive them for storage in liquid nitrogen must be prepared in advance. We are using straws (in the field known with a French word: paillettes) from IMV (IMV, Cassou straws, France, about 13 cm long, a diameter of 2 mm and a very thin plastic wall of about 0.1 mm; they can be obtained from Albrecht GmbH, 88326 Aulendorf, tel. 07525/20572). There are identification sticks, useful for capping the straws and for writing information about the embryos, available from the same company. These straws are open from one side and have a stopper in the other side. This stopper allows air to be sucked with a syringe in order to fill the straw with liquids, but polymerizes once it gets in direct contact with a liquid, sealing the capillar at this end. The other end will then be closed with the identification stick. 

	This figure above represents the straw. In A, it is displayed an already filled straw, with the distribution of the buffers. In B, it is presented an empty straw with some marks used for loading the buffers.

1) Prepare the straw as follows. Aspirate Vitrification Medium (VM) until it reaches a level between marks B and C (as indicated in the figure). Do not cross the C border with VM.

2) Aspirate a small air bubble (from the open end until the mark labelled with A). The straw is ready-to-use. Leave it near you and now concentrate on the embryos.

3) On a empty Petri dish, place rows of drops with Equilibration Medium (EM). Each drop should be about 100-200 μl. You will need 5-7 drops/row. Each row will be used only once and will represent the embryos which you will freeze in ONE straw. Place some additional drops of EM, to clear and equilibrate your glass pipette.

4) Transfer the embryos (kept in M2 medium) into the first drop of a given row. Have a running clock in front of you. The embryos now will be transferred (with the help of the pipette) from drop to drop, in the same row, in order to equilibrate them in this first buffer. The whole process must not take more than 10 minutes, with a minimum time of 7 minutes, at room temperature. Spend 1-2 minutes/drop and do a couple of washes (pipette in and out) in each drop to promote the equilibration with the buffer.

NOTE: This contact of the embryos with EM results in the first step of dehydration. During the first 1 or 2 minutes the embryos will collapse and decrease the inner volume. This is because water will have an initial tendency to leave the embryos, which are less concentrated than the surrounding buffer (there is less water in the EM buffer than inside the embryos). After these first minutes the second osmotic forces will take over. Glycerol is more concentrated outside than inside the embryos. Therefore, glycerol, at moderately slow speed (lower than the speed of water coming out) will have a tendency to diffuse into the embryos until the equilibration point is reached. This second part will take about 7 minutes and, at the end, the embryos should have recovered from the collapse. Not all embryos will resist this osmotic stress. Damaged embryos are more likely to die in this first step. This normally will represent a small proportion of the embryos (less than 10%). Those embryos which, at the end of these 10 minutes in EM, have not recovered fully the initial volume have to be discarded for further manipulation.

	Transfer between 5-10 embryos each time. It is better to split the embryos in as many straws as possible rather than concentrated them in a few straws, with higher risks of looses if problems are found with an individual straw. Using a small number of embryos helps in quickly localizing in the consecutive EM drops and speeds the whole process. Keep in mind that embryos, at the beginning, are less heavier than the medium and will have a natural tendency to float and accumulate on the surface of the drop. As the treatment progresses (from drop to drop) they will equilibrate with the EM and accumulate on the surface of the plate.

	With practice, different groups of embryos can be started at overlapping times in parallel rows of drops. To begin with, it is highly recommended to proceed one straw after the other.

5) Prepare a small Petri-dish (3 cm of diameter or so) filled with a thin film of VM buffer (about 2-3 mm high, not more). Wash you pipette in an eppendorf tube containing VM. Keep all your embryos grouped at the centre of the last EM drop. Come in with the pipette loaded with VM and make a circle around the embryos, as close as possible, by releasing a bit of the VM buffer. 

NOTE: Surrounding the embryos with a thin circle of VM buffer will avoid the natural tendency they will have to escape from a heavier buffer (VM is heavier and more viscous than EM). 

6) Try to collect all embryos in a very small volume and transfer them into the small Petri dish with VM. Try to maintain all embryos grouped in order to facilitate recover of all of them in the smallest volume as possible.

NOTE: This will be the second (and more intense) step of dehydration. VM buffer is more toxic than EM for the embryos. We should try to work as quick as possible, to minimize the exposure of embryos to VM at room temperature.

7) Finally, take the open end of your prepared straw (from step 2). Make sure the air bubble has the right size. Go with it near the embryos and aspirate (now use the syringe connected to the stopper) them into the straw in the smallest volume as possible. The volume of VM (with all embryos) should be between marks B and C of the straw.

NOTE: It is better to approach the embryos from one side with the open end (rather big under the microscope) of the straw than trying to go directly above them (which will disperse the embryos in all directions and require more volume, and time, to aspirate all of them). 

8) Quickly, load a second air bubble in the straw (again until the mark labelled with A, as indicated in the figure) and, immediately, load the straw with Dilution Medium (DM) which you will have prepared in an open eppendorf. Aspirate DM until the first volume of VM (without embryos) reaches the stopper (the contact of liquid and a substance present in the stopper will seal this end of the straw).

NOTE: The drop of VM with embryos, surrounded by two bubble airs will travel along the straw while you aspirate the DM medium. With this strict order for loading buffers you avoid contact of the drop containing the embryos in VM with any other buffer (other than the previous drop, travelling ahead, which also contains VM and "cleans" the inner walls of the tube). 

The column of DM buffer (much larger in volume than the small drop containing the embryos) does not have glycerol and will serve as an immediate diluent for the embryos after thawing. It contains a high percentage of sucrose, to avoid explosion of the embryos by the sudden contact with a buffer with no glycerol in it. The water would have an automatic tendency to penetrate and blow the embryos away. The presence of sucrose outside will prevent this natural osmotic force from acting (there is not much water also outside) and will allow enough time for the embryos to discard the glycerol present inside and substitute it, slowly, by water, without exploding.

9) Fix firmly the identification stick (write on it the number, strain of the embryos and the date; use pencil or special pen, normal pens disappear under liquid nitrogen) to close the open end of the straw and quickly introduce the straw (handed from the identification stick) into a liquid nitrogen bath. First, introduce the stopper part of the straw, until the surface of the liquid nitrogen reaches the place where the second air bubble is located. This will keep the two drops of VM (the second one with the embryos) submerged in the liquid nitrogen. 

NOTE: If everything went OK you should NOT observe crystal formation in the VM drops, which should remain clear and transparent during ALL the process. This is what is called "vitrification", opposite to crystallisation or ice formation.

10) As the temperature begins to low along the straw you will start seeing crystal formation (the liquid becomes opaque and milky) in the long DM drop. Submerge the straw inside the liquid nitrogen trying to keep the growing crystallisation front of the DM just above the surface, until it reaches the end of the straw. Finally, release the straw inside the flask with liquid nitrogen.

NOTE: Sometimes, due to changes in volume associated with temperature, the identification stick comes off from the end of the straw. You can always take out the upper part of the straw, warm it until it thaws, and fix again the stick tightly. 

Once the embryos (always in the opposite end from the identification stick) have entered the liquid nitrogen must be INSIDE liquid nitrogen (liquid phase, not nitrogen vapours!) during all time to guarantee full recovery and permanent storage.

All manipulations from releasing the embryos in the Petri dish with VM (step until totally introducing the straw in the liquid nitrogen (step 10) must be performed as quick as possible (1 minute or so). Prepare your self before starting this final and crucial step.

11) For proper storage of the straws use gobeletts (Plastic cylinders with a closed end, available from Albrecht GmbH) which will fit in the metallic containers in which the straws will be stored inside the big liquid nitrogen tank. These metallic containers are available from Schneider, Metallwarenfabrik, Nägeleingasse 20, 91413 Neustadt/Aisch. The gobelets are very helpful because they will keep liquid nitrogen inside the metallic containers when you pull them out of the tank, briefly, to look for particular embryos. In this way, embryos will always be kept UNDER liquid nitrogen. The metallic cylinders have, otherwise, a grid on the bottom which would empty after pulling them out of the liquid nitrogen and will momentaneously increase the temperature of the straws, affecting to the viability of the embryos.

THAWING

1) Locate the desired straw and transfer it, quickly, to a portable liquid nitrogen container. Make sure all straw is covered by liquid nitrogen.

2) Prepare a beaker with about 2-3 litres of water, at room temperature.

3) Take the straw (from the identification stick) and put it, directly and as quick as possible, inside the water of the beaker. Shake it carefully and in 5-10 seconds the long DM drop will thaw (will become transparent). During all the process monitor the two drops of VM. They should remain transparent all the time.

4) As soon as the DM is totally thawed, take out the straw and shake it, firmly and quickly (supporting from the end of the straw capped with the identification stick), to promote immediate mixing of DM with the two DM drops.

NOTE: The sudden mixture of DM and VM will immediately dilute the glycerol in which the embryos were cryopreserved and start the osmotic exchange between glycerol and water, inside the embryos.

5) Wipe out the water from the outside surface of the straw and cut the cotton plug of the straw as close as possible to the side of the stopper near the inner buffer of the tube.

6) Remove the identification stick and connect a syringe. Flush the totality of the straw in a single drop on a Petri dish. Before, on the same Petri dish, you have prepared a row of drops with DM buffer, for a series of washing-dilutions (from drop to drop) in a manner similar to the protocol described in the freezing steps.

7) Using the pipette and the microscope try to localize all embryos contained in that straw. The embryos have a tendency to accumulate around the periphery of the drop. Transfer all embryos to the first drop of fresh DM medium and pipette up and down to increase buffer exchange. Transfer the embryos from drop to drop. Use about 5-7 drops. The process should take between 8 to 10 minutes. Prepare a running clock in front of you.

NOTE: If you find, at the first look, less embryos than expected then aspirate and re-flush once or twice the straw. Normally, by repeating flushing the rest of the embryos will come out. Embryo recovery, at this point, should be greater than 90%.

8) After 8-10 minutes in different drops of DM transfer all embryos to a Petri dish with HB1 medium (HB1), at room temperature, and allow them to recover for 20-30 minutes.

NOTE: It is very important to allow recovery at room temperature. Do not incubate at +37^oC!.

9) Finally, transfer the embryos into a new Petri dish with M2 medium and carefully look for their morphology. It should be very clear those embryos which look almost identical as before being frozen from those displaying different levels of damage (collapsed, wrapped, black or with brown inclusions, with death cells, etc...). Morphological recovery, at this point, should be greater than 75%.

10) This protocol can be done at overlapping times with different straws and the embryos can be collected in the HB1 or M2 media. Select about 10-20 embryos per group and load them in transfer capillars, as discussed in section 2.e.

11) Transfer the embryos back into the oviduct of pseudopregnant recipient females from day +1 (from the day BEFORE of the actual day in which you are thawing the embryos). Expect a potential delivery about 18 days later.

NOTE: Pregnancy ratios might vary a lot. However, the efficiency, with this technique, is higher than with other methods. With practice, around 30% of transferred embryos should implant and resume embryo development, resulting in newborns. About 100-200 embryos must be kept frozen, from a given line, to guarantee recovery of the line. That represents between 10-30 straws. Thaw one or two straws from each batch, the day after freezing or so, to verify that everything went OK. The most important requirement for this technique is the necessity of maintaining the embryos ALL THE TIME under liquid nitrogen. Make sure there is always enough liquid nitrogen in your tank to avoid potential problems. It is a good tip to split the number of straws in two groups and, if possible, keep them frozen in separate liquid nitrogen containers, to minimize all risks.

	Solutions:

	Vitrification medium (VM)			mg/100 ml

	NaCl						800
	KCl						20
	CaCl_2-2xH_2O					1
	KH_2PO_4					12
	MgCl_2-6xH_2O					5
	HEPES						476.6
	Glucose						100
	BSA						75

	Glycerol (from >99% pure stocks)			47.5 ml (each 100 ml of VM)
	Polyethylenglycol (PEG 8000)			6 g (each 100 ml of VM)

	Prepare it with distilled sterile H_2O. Filter through 0.22 μm to sterilise. 	
	Store it for some months at +4^oC.

	HB1 medium (HB1)					mg/100 ml

	NaCl							800
	KCl							20
	CaCl_2-2xH_2O						12
	KH_2PO_4						13.6
	MgCl_2-6xH_2O						10
	Na-pyruvate						3.6
	HEPES							476.6
	Glucose							100
	BSA							300
	Penicillin-G (Na salt)					6

	Prepare it with distilled sterile H_2O. Filter through 0.22 μm to sterilise. 	
	Store it for some months at +4^oC.

	Equilibration medium (EM)			For 100 ml:

	25% VM					25 ml VM
	75% HB1					75 ml HB1

	Prepare it from sterile stock solutions. Store it for some months at +4^oC.

	Dilution medium (DM)				For 100 ml:

	1 M sucrose (in HB1)					34.230 g
	HB1							up to 100 ml

	Filter through 0.22 μm to sterilise. Store it for some months at +4^oC.