WO1999011121A1

WO1999011121A1 - Method and auxiliaries for cryopreservation of biological material such as egg cells

Info

Publication number: WO1999011121A1
Application number: PCT/DK1998/000375
Authority: WO
Inventors: Gabor Vajta
Original assignee: Danish Institute Of Agricultural Sciences
Priority date: 1997-09-03
Filing date: 1998-09-03
Publication date: 1999-03-11
Also published as: AU8852098A

Abstract

In the cattle breeding sector it has been known for several years that it is possible to preserve egg cells or embryos by introducing them into an end of a narrow tube or 'straw', which is thereafter subjected to cryogene cooling. For some species such as pigs this preservation, however, has failed to work in practice, and the invention provides for improved results in prescribing the use of still narrower tube ends which can easily, by capillary action, be loaded with a small amount (1-2 νl) of a holding liquid for the item to be cooled, and which, when dipped into the cryogene cooling medium, provides for a close cooling contact with this medium, partly through an open end connection and partly through an outer pipe end wall of a very small thickness. A high preservation quality is achieved by the associated extremely fast cooling. Preferably, the thin tube ends are provided by a pulling out of the middle portion of a thermoplastic 'mini straw', and then cutting the straw at the middle of the resulting narrowed straw length, thus leaving the opposed thicker pipe ends as convenient handle portions.

Description

Method and auxiliaries for cryopreservation of biological material such as egg cells.

The present invention relates to a method and auxiliaries for cryopreservation of biological material such as fertilized or non-fertilized egg cells by vitrification for later implantation, whereby the cells, isolated in a holding liquid, are caused to be introduced into an end portion of a narrow carrier tube, this end portion thereafter being sub- jected to cryogene cooling, preferably in being dipped into liquid nitrogen at -196°C. After some time of storage and/or transportation in this supercold condition, the cells (oo- cytes or embryos) are thawn by dipping the tubes in a physiologically tempered liquid, and they are then removed from the tubes for further processing. Methods of this type are disclosed in US-A-3, 354,470 and 3,866,598.

The narrow carrier tubes, commonly called "French mini straws" are made of a thermoplastic material, very typically with an inner diameter of 1.7 mm and a wall thickness of 0.15 mm. With reference to their internal volume they are of a length making it natural to classify the "mini straws" by the term "0.25 ml straws". With reference to their internal volume they are of a length making it natural to classify the "mini straws" by the term "0.25 ml straws". With adaptation to their small diameter there has already been developed various handling equipment and even identification printing equipment for facilitating the use of the straws, as well as equipment for subjecting the straws to the required suction up of the cell holding liquid and for closing the straw ends to prevent a fall-out of the liquid.

For a row of years this technique has been used successfully in the cattle breeding area, but it has been found that there are some relevant areas in which it has not proved applicable, e.g. already in pig breeding, where very high death-rates of embryos have been registered, without giving rise to specific explanation of reasons for this fact.

In connection with the present invention it has been found, surprisingly, that the same technique, only slightly modified, is in fact usable for achieving highly promising results for the pig breeding sector and even other difficult sectors. The required modification, basically, is the use of considerably still thinner tubes or tube ends for holding the cells, this concept having a row of important consequences as explained in more detail below.

In practice, what has been found required is to work out a deformation of the existing mini straws by heating their middle portions and expanding their length so as to pull out the softened middle portion into a thinner tube portion, with an inner diameter reduced to some 0.8 mm and with the wall thickness reduced to some 0.07 mm. The pulled out tube is cut with a sharp cutter at the thinnest point and is thus converted into two usable straw units. This conversion offers dramatic changes in the operational conditions of the straws, and in view of the achieved good results it is reasonable to assume that the keyword of the success is "rapidity" of the cooling and/or the warming process. Of course, the invention will apply to the use of any kind of such a narrow tube or tube end, no matter how it is produced, when only it has a very low heat insulation capacity. The said important consequences can be listed as follows:

1) The said holding liquid will typically have a viscosity close to that of pure water, and when the inner tube diameter subcedes 1 mm the tube will exhibit capillary effect, whereby the holding liquid with the eggs, e.g. deposited in a droplet on a carrier plate, will rise into the tube without any need of applying suction equipment for the purpose. The natural rise of the liquid is fully sufficient to bring one or more eggs into the tube end.

2) Upon the said rise of the liquid with one or more cells into the tube end there will be no need to effect a closing of the tube end in order to prevent an outflow therefrom, as the liquid will be held back already by the capillary effect itself.

3) The amount of capillarily loaded liquid will be very much smaller than the volume hitherto used for the suction in of a relevant tube filling. In connection with the invention, a liquid volume of some 1-2 μl will be sufficient, which is far below the traditional volume . 4) With the combined use of a very small amount of liquid and a very small thickness of its surrounding tube wall, this will naturally result in a highly efficient and rapid cooling of the liquid when the tube end is immersed in a cryogenic cooling medium. 5) Inasfar as the tube end does not need to be closed in order to hold back the cell carrying liquid, the cryogene cooling medium may act directly onto the surface of the carrying liquid at the end of the tube, thus enhancing the cooling action considerably. 6) The same, of course, will apply to the later warming, where a very rapid heat transfer will be secured. As soon as the holding medium returns to its liquid state, the thawing liquid may enter the tube end, but the eggs will sink slowly down and be delivered from the tube end all by themselves, without the use of auxiliaries of any kind.

7) It is acknowledged that it is required to make use, in the said cell holding liquid, of certain cryprotective agents in order to limit the potential damages to the cells by the fast cooling itself. These agents happen to be directly toxical to the cells, and with the invention it is possible to increase the concentration of these agents because of the achievable very fast cooling of the cell carrying liquid. Such an increase may very well contribute to the improved result.

8) It has been found that superficial damages on the cells are liable to occur in particular in the cooling range between +10°C and -5°C, due to the behaviour of intracellular lipid droplets, and it seems that these damages are greatly reduced when the cooling/ -warming takes place at the highly advanced speed as conditioned by the invention. 9) The method does not require highly skilled practitioners, as it is very easy to carry out. 10) With the use of the preferred embodiment of the tubes, viz. the conventional "mini straws" being pulled out at one end, the remainder of the tube or straw will be unchanged, whereby the tube can still be handled by the already existing handling equipment, including the equipment for identification printing of the tubes.

At the scientific level there are several other aspects to be discussed, but in the present connection it is considered sufficient to note that the use of the narrowed holding tube implies a whole row of important advantages. The narrowness of the tubes is well defined by their ability to perform capillary attraction of the cell carrying liquid, and this by itself seems to be new in the art.

Admittedly, while it is held to be important that the cooling liquid can act directly on the egg holding liquid in the open tube end, this also implies some moments of risk, although rather remotely. For one thing, the cooling liquid could be somehow biocontaminating, and for this reason it may be prescribed that only sterilized cooling liquid be used. For another thing, though hardly thinkable, some kind of infectious matter could spread through the cooling liquid and intrude into the frozen egg holding liquid in the tube end or intrude from the outside into the broader outer end of the tube. In fact, this outer end could be closed even beforehand, as the capillary suction-in of 1-2 μl would still be possible despite such an outer closure of the tube. However, in order to provide for a tight closing of the tube at both ends thereof, according to the invention, it is preferred to arrange for the tube, after the end cooling thereof, to be encapsulated in a surrounding, wider tube structure that is closed at both ends. Thus, once the re- quired cooling has been effected at the narrow tube end, the tube may be briefly retracted from the chilling liquid and inserted into an outer tube casing e.g. having a rear end closed beforehand, while its open front end is caused to be closed immediately upon the introduction of the egg carrying tube, e.g. by heat sealing.

Such a heat sealing, of course, should be effected both rapidly upon the egg carrying tube and being lifted off from the cooling liquid and at a safe distance from the tube end, e.g. 1 cm. Preferably the outer tube should be precooled. Immediately after the said closing, the set of tubes is replaced in the chilling liquid. Alternatively the outer tube may be preclosed at the end adapted to house the said narrow tube end, whereby the rear end of the outer tube is the one to be finally closed.

It should be mentioned that the said narrow inner diameter of approximately 0.9 mm provides for a natural capillary holding liquid intake corresponding to a liquid column of some 10 mm. Of course, one or more eggs could well be held in a lower column, i.e. in a slightly wider space, but it is feared that a liquid column of only a few mm will not be stable when brought into contact with the cooling medium; it could even be retracted from the tube end, and such problems have not been observed in connection with columns of 8-12 mm. Even a still narrower space could be used, for the formation of liquid columns of more that 12-15 mm. This, however, would hardly be of any practical interest, and at least it would not enhance the rapidity of the freezing of the holding medium.

Care should be taken that the egg holding droplets used for the loading of the thin tube ends should not be much larger than what is required for the said capillary intake, that is a volume of 1-2 μl . Once the tube end gets in opera- tive touch with the droplet, breaking the surface tension thereof, the capillary intake happens very fast, and the operator will not have time to "chase" the one or more egg cells or embryos in the droplet. If they are not introduced by the rapid natural intake it will be unrealistic to seek to load them afterwards.

According to the relevant prior art, a complete vitrification (fast freezing) operation takes some 4 minutes, of which some 2 minutes are used for the very cooling. With the invention the complete operation can be done in 1-2 minutes, typically some 1 minutes, with the cooling accomplished during a few seconds or less, i.e. practically momentarily. In other words, while the cooling rate with the relevant prior art is reported to be some 2,500°C/min, the corresponding figure in connection with the invention has been measured to be, representatively, some 22.5000°C/min. , i.e. close to ten times as fast. For the sake of completeness it should be mentioned, however, that this figure refers to the temperature interval between -25 and -175°C, while the corresponding figure for the interval between zero and -195° was only 16.700°C/min. , yet still very high.

In the following the invention is described in more detail with reference to the drawing, in which Fig. 1 is a schematic view of a straw end in a ready-to- be-filled position,

Fig. 2 is a corresponding view of the straw end dipped in a cryogene freezing medium,

Fig. 3 is a similar view of the straw end during thawing, and

Fig. 4 is a schematic view of the entire straw enveloped in a surrounding tube member.

Briefly, Fig. 1 shows that a small droplet 2 of a holding liquid, inocculated with cell formations (oocytes or embryos) 4, is placed on a carrier surface 6, and that the droplet is approached by the mouthing of a thin tube portion 8 projecting from a wider tube portion 10. When the tube end effectively engages the droplet 2, the same and its contents will rise into the narrow tube portion by the capillary effect thereof, to a level 12 therein.

Immediately thereafter the tube portion 8 is plunged into a cryogene bath 14, Fig. 2, in which the holding liquid will freeze almost instantaneously due to 1) the very thin wall of the tube portion 8, 2) the very small volume (1-2 μl) of the holding liquid, and 3) the direct contact between the cooling medium 14 and the front end of the holding medium column 16 in the tube end. endwise into a warming liquid 18, Fig. 3, whereby the frozen holding medium in the column 16 is rapidly thawn, and the cell formations 4 simply slide down into the warming liquid 18, from where they are picked up for further processing. As already mentioned, it is preferred to encapsulate the tube or straw 8, 10 in an outer casing of a wider tube member 20, Fig. 4, as soon as a first dipping in the bath 14 has been completed. This outer tube may be preclosed at one end and afterwards closed at the opposite end, and preferably it may be precooled before the insertion of the tube member 8, 10. After the ultimate closing, the combined structure 20, 8 and 10 is returned to a position dipping into the bath 18. For the warming stage, the outer tube 20 is cut open, preferably while still in contact with the cooling medium, e.g. in the vapour area just above the liquid, and immediately thereafter the inner tube is taken out and transferred to the warming liquid.

It should be emphasized that the invention is not limited to the handling of eggs or embryos, as there is reason to believe that in particular the easy handling and the very fast cooling/warming will be advantageous in connection with cryopreservation also of other items, e.g. tissue pieces.

By way of example, the following experiment should be mentioned:

Example:

Embryo production:

Except where otherwise indicated, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA) . Embryo production : The methods used for oocyte recovery, in vi tro maturation, fertilization and embryo culture have been described previously (11) . Briefly, oocytes were aspirated from slaughterhouse-derived ovaries and matured in TCM-bicar- bonate (TCM-199 medium (Gibco BRL, Paisley, UK) containing 25 mM sodium bicarbonate, 0.2 mM sodium pyruvate, 0.4 mM 1-glu- tamine, 50 mg/ml gentamycin, adjusted to pH 7.4 and 280 mOsm supplemented with 15% calf serum (National Veterinary Laboratory, Frederiksberg, Denmark) under paraffin oil at 39°C in 5% C0₂ in humidified air. Frozen-thawed, Percoll-selected sperm was used for in vi tro fertilization (Day ()) . Presumptive zy- gotes were cultured in TCM-bicarbonate supplemented with 5% (10% from day 5) calf serum on granulosa cell monolayer. All cultures were performed at 39°C in 5% C0₂ in humidified air.

The open pulled straw (OPS) method : All manipulations were performed on 39°C heated stages at a 25-27°C room temperature. Day 2 (8 cell stage) , Day 3 (16 cell stage) and Day 4 (early morula stage) good quality embryos were transferred to the holding medium (TCM-199 medium containing 2.5 mM Hepes, 5 mM bicarbonate, 0.2 mM sodium pyruvate, 2.5 mg/ml amphotericin B and 15 IU/ml herapin, adjusted to pH 7.4 and 280 mOsm) supplemented with 20% calf serum.

French mini-straws (250 μl , LMV, L'Aigle, France) were heat-softened over a hot plate, and pulled manually until the inner diameter and the wall thickness of the central part decreased from 1.7 mm to approx. 0.8 mm, and from 0.15 mm to ap- prox. 0.07 mm, respectively. The straws were cooled in air, then cut at the narrowest point with a razor blade. In order to vitrify, embryos (4 to 6 per group) were initially placed for 2 min in holding medium containing 10% ethylene glycol and 10% dimethylsulphoxide for 2 min. Subsequently, the embryos were transferred between three 8 μl droplets of 20% ethylene glycol, 20% dimethylsulphoxide and 0.6 M sucrose dissolved in the holding medium for approximately 10 s each. Loading was performed by placing the narrow end of the pulled straw in the third droplet and aspirating embryos in a 2 to 3 mm long liquid column (1 to 1.5 μl volume) using the capillary effect. Subsequently the straws were immediately submerged vertically into liquid nitrogen.

At warming, the open end of the straw was immersed vertically into 1.2 ml holding medium containing 0.3 M sucrose. The vitrification medium became liquid in 1-2 s, whereupon the holding medium entered the straw. Immediately afterwards the embryos gradually descended out of the straw into the culture dish. Forty to 50 s after warming, the embryos were transferred into 1.2 ml holding medium with 0.15 M sucrose, then twice into 1.2 ml of holding medium, for 5 min each. Finally, the embryos were placed into the original culture dish on a granu- losa cell monolayer and cultured in 400 μl TCM-bicarbonate supplemented with 10% calf serum under paraffin oil at 39°C in 5% C0₂ in humidified air. Control vi trification in original straws : Embryos were selected and incubated with the diluted and concentrated vitrification solutions as described above. A 250 μl French straw was loaded by syringe aspiration with 180 μl holding medium containing 0.3 M sucrose, then with three droplets of the con- centrated vitrification solution, separated from each other and from the holding medium by air bubbles . The second droplet of the vitrification solution contained the embryos. The straws were heat-sealed to avoid outflow, and slowly submerged vertically into the liquid nitrogen, with the droplets of vit- rification solution being submerged initially. The time required for loading, sealing and submerging was 20 to 30 s.

The straws were warmed first for 8 s in air, then submerged horizontally into a 39°C water bath until the holding medium had reached the liquid phase. The straws were then held by hand at the sealed end and shaken three times in order to mix the liquid columns, and the contents were then expelled into 100 μl of holding medium containing 0.3 M sucrose for 1 min. The subsequent rehydration steps and embryo culture were performed as described above. Evaluation : Zona fracture following rehydration was examined under a stereomicroscope . On Day 8, the number of blasto- cysts was determined under a stereomicroscope. Data were analyzed by the chi-square test with a P<0.05 considered to be statistically significant.

RESULTS

As presented in Table 1, no embryos survived vitrification in the control experiment using normal-size sealed straws. Using the OPS method, the highest survival rates were achieved with the embryos vitrified on Day 4 (Fig. 1) . However, the blastocyst per oocyte rates were not different between embryos vitrified on Day 2,3 or 4. The frequency of zona fracture damage was 22/70 (31%) and 3/212 (1%) in embryos vitrified in the normal size straws and with the OPS method, respectively (P<0.05).

Table 1. Results of the vitrification experiments.

Treatment Oocyte Vitrif:ied Blastocyst Blastocyst/ Blastocyst/

Values in the same column with different superscript letters are significantly different (P<0.05).

It will be appreciated that the invention, in addition to its primary aspect of handling and storing biological items in connection with cryogene preservation, provides for an easy manner of handling even other tiny objects in connection with other kinds of treatment, e.g. irradiation or just transportation.

Claims

C L A I M S :

1. A method of handling small items for treatment, in particular for cryopreservation of biological items such as embryos or fertilized or non-fertilized eggs, whereby the item is deposited in a droplet and is caused to be introduced, together with droplet liquid, into a thin carrier tube by capillary intake therein, whereafter the tube is used as a holder for holding the item in droplet liquid during the relevant treatment.

2. A method according to claim 1 for cryopreservation of egg cells and the like introduced in a holding liquid comprising a cryoprotecting agent, whereby the thin carrier tube is held against a cell containing droplet and, immediately upon the capillary loading of the droplet into the tube end, dipped into a cryogene bath with the thin tube end in non- closed condition.

3. A method according to claim 2, wherein the tube end portion is made of a thermoplastic material and has a wall thickness of less than 0.1 mm.

4. A method according to claim 2, wherein the volume loaded into the tube end is 1-2 ul .

5. A method according to claim 2, wherein the naturally shaped liquid column in the thin tube end is at least 5 mm, preferably 8-12 mm long.

6. An auxiliary for use with the method according to claim 1 and constituted by an item holder having at least one protruding tube portion with an inner diameter of less than

1 mm, preferably 0.6-0.9 mm.

7. An auxiliary according to claim 6, in which the holder is a tube having said protruding tube portion at one end and a remaining tube portion of increased diameter.

8. An auxiliary according to claim 7, in which the thin tube end is a portion pulled out from the end of said remaining tube portion, the tube being made of a thermoplastic material.