EP0304358B1 - Storing tube for small quantites of substances, especially biological ones, and its use in a fertilisation process - Google Patents

Description

"straw for the storage of small quantities of substances in particular biological and its use in a process of fertilization."

The present invention relates to a flake of the kind used for the storage of small quantities of substances, in particular biological substances, consisting of a segment of tube the two ends of which respectively define a first and a second end zone, the first end zone being equipped with a corresponding plugging means, said first.

As described for the first time in patent FR-995878 (CASSOU), such a flake is in the form of an elongated thin tube equipped, in one of said end zones, with a tripartite plug comprising two buffers of a fibrous material permeable to gases and which encloses a powder initially also permeable to gases and capable of transforming on contact with an aqueous liquid into an impermeable paste or gel by creating a sealed stopper which can nevertheless slide in the tube.

The other end zone of the tube segment normally remains open for the introduction of the liquid to be preserved, generally by suction exerted through the tripartite plug.

• obturation par bouchage,
• soudage, par exemple aux ultrasons,

   application de la technique décrite au brevet précité consistant à tremper l'extrémité à obturer dans une poudre semblable à celle utilisée dans le bouchon tripartite.To protect the loaded liquid in the tube from contamination and vice versa, if it is a partially dangerous liquid for the environment, to avoid its diffusion out of the straw, the second end zone says naturally to be closed and until now there were various possibilities:

• plugging,
• welding, for example ultrasonic,

application of the technique described in the aforementioned patent consisting of dipping the end to be closed in a powder similar to that used in the tripartite cap.

Patent application DE-A-2 506 108 (POPOVIC) describes a flake plugged in one of its two end zones by another tripartite plug initially impermeable to gas but capable of forming a tight piston.

This tripartite plug has instead of pads made of a gas permeable fibrous material of the CASSOU cap, pads of a gas impermeable polystyrene foam, enclosing a powder similar to that of the CASSOU cap liquids are introduced into this flake by translation of the tripartite plug from one end to the other.

As in the French patent above, after filling, the second end region of the straw should be closed.

Said polystyrene foam pads have the serious drawback of considerably reducing their volume at the temperature of liquid nitrogen and of transforming into a layer of material which no longer closes the tube. Only the gel from the powder then ensures the separation of the interior of the straw with the exterior.

Due to the increasingly frequent needs for handling and storage of particularly dangerous biological samples, the need appeared to reinforce protection against any risk of contamination of operators and the environment.

The present invention relates to the creation of a straw offering high protection against any risk of contamination while being suitable for simple, convenient and safe handling.

The present invention thus relates to a straw for the storage and handling of small quantities of biological substances, composed of a rectilinear segment of tube made of transparent polymer material comprising at its two ends first and second end zones equipped with plugging means respective watertight, characterized in that these sealing means each consist of a self-sealing compact elastomer plug, called ECA, capable of sealingly adhering to the internal wall of the tube and allowing a hollow needle to pass through it, the tightness being maintained both around the needle in place and in the plug after removal of that -this.

Thus the straw according to the invention, when it is empty of any substance, already has at these ends first and second sealing means constituted by the two plugs of self-sealing elastomer. To introduce a substance inside this flake, it is necessary to pierce the said self-sealing plug, which will be referred to below as ECA with a hollow needle whose conduit allows the passage of the chosen substance, the latter having to be sufficiently fluid to not not cause the obstruction of said duct. The withdrawal of the needle causes the filling, thanks to the elasticity of the elastomer, of the channel created by the needle in the stopper. The two self-sealing plugs allow you to inject and remove any fluid from either end, by inserting a needle into each of the plugs.

Thus, such a straw can be handled in an automated manner and the delicate operation of the final plugging, when the straw contains a dangerous product is avoided.

Another object of the present invention relates to the use of a straw, where the first as the second sealing means is constituted by an ECA plug, in a process the in-vitro fertilization of at least one female gamete by a gamete male from a group of male gametes, the gametes being of human or animal origin.

This method consists in forming in the tube segment a plurality of contiguous liquid segments respectively adapted to constitute on either side of an intermediate connection zone, a zone of loading of male gametes near one of said terminal areas and a loading area of female gametes near the other terminal area.

This process has the advantage of being biomimetic, in that the sperm have to travel a certain distance in a small diameter tube before waiting for the oocytes to ensure fertilization. The oocytes are reached first by the most mobile and vigorous sperm.

This in vitro fertilization is naturally carried out at an appropriate temperature which varies according to the species concerned.

• la figure 1 représente en coupe axiale partielle une paillette de l'art antérieur décrite dans le brevet FR-995 878 ;
• la figure 2 représente en coupe axiale partielle une paillette de l'art antérieur décrite dans la demande de brevet DE-A-2 506 108 ;
• les figures 3 à 6 représentent chaque fois en coupe axiale partielle des modes de réalisation d'une paillette selon la présente invention ;
• la figure 7 représente une vue schématique d'un appareil destiné à la fabrication des paillettes représentées aux figures 4 à 6.

Other advantages and characteristics of the invention will also emerge from the description which follows, by way of examples, with reference to the appended drawings in which;

• Figure 1 shows in partial axial section a straw of the prior art described in patent FR-995,878;
• 2 shows in partial axial section a straw of the prior art described in patent application DE-A-2 506 108;
• Figures 3 to 6 each time show in partial axial section embodiments of a straw according to the present invention;
• FIG. 7 represents a schematic view of an apparatus intended for the manufacture of the flakes shown in FIGS. 4 to 6.

The straw of Figure 1 consists of a segment of cylindrical tube 1 open at its two ends 2, 3. These define respectively a first and a second end zone.

The first end zone contains, inside the tube 1, a tripartite plug 4.

This stopper 4 is constituted by a segment 5 of a special powder between two pads 6, 7 of a fibrous substance permeable to the fluid which is presented here in the form of a braided wick. The special powder of segment 5, initially porous to gases, has the property of rapidly transforming on contact with an aqueous liquid into an impermeable paste or gel which adheres to the wall of the tube and hardens on contact with air, thus forming an airtight stopper. to fluids. The prior art tube thus has only one stopper. The filling of the segment 8 of the tube 1 with an aqueous liquid can only take place through the open end 3 because of the very function of the tripartite plug.

The straw of FIG. 2 also includes a tripartite plug 104 in a first end zone. A segment 5 of special powder is inserted between two pads 106, 107 of polystyrene foam. The quality of the foam gives this tripartite stopper 104 a function different from that of the tripartite stopper 4 in FIG. 1.

In fact, at ambient temperature, this stopper 104 is gas tight and essentially serves as a movable piston inside the straw.

At the temperature of liquid nitrogen, the cells collapse on themselves with a significant reduction in volume of the buffers 106 and 107, only the powder of segment 5 more or less solidified then intervenes between the content of the glitter and the exterior. It should also be noted that polystyrene is not an elastomer.

FIG. 3 represents a straw according to the invention which, compared with the straw of the prior art, further comprises towards the edge of the first and second ends 2 and 3 of the tube 1, inside, plugs 9 made of compact elastomer . This stopper has a length limited to a small fraction of the total length of the tube, for example one twentieth of it.

This plug 9, called ECA plug, consists of a compact elastomer which adheres to the inner wall of the tube and which has the property of being passed right through by a hollow injection needle by creating a sort of channel which grips the needle body tightly. This momentary drilling allows communication the inside of the tube 1 with the outside via the plug 9 made of compact elastomer, and therefore of carrying out material transfers via the needle duct between the inside of the tube and the outside. When these transfers are complete the hollow needle is withdrawn and the compact elastomer is sufficiently elastic so that the passage channel of the needle closes hermetically. Advantageously, the compact elastomer will have a final Shore A hardness of between 20 and 50.

By compact elastomer is meant an elastomer without a cell, so as to avoid collapse and shortening of the plug at cryopreservation temperatures.

The compact elastomer can be a thermoplastic rubber chosen from the cariflexes TR (registered trademark of the Shell company) Solprene (registered trademark of the Philips company). Europrene Sol T (registered trademark of Anic) or a one- or two-component elastomer, vulcanizable at room temperature like Rhodorsil translucent silicone sealant (registered trademark of Rhône-Poulenc Corporation), Silopren (registered trademark of Bayer Company), Pourable Silicone (registered trademark of Rezolin Company) or Silbione RTV (Rhône-Poulenc) with two components.

The elastomers are chosen to be non-cytotoxic and with no salting-out or absorption properties with respect to the biological medium introduced into the tube.

The plug 9 made of compact elastomer can be placed inside the tube by conventional plugging, using a preformed plug, by force-fitting inside the tube 1. The tube is held during this operation in a support barrel which encloses it tightly to avoid any deformation or buckling.

Another preferred method consists in injecting inside the tube towards its end 3 a metered amount of a thermoplastic elastomer or vulcanizing cold.

Unlike flakes which have a tripartite cap, it is possible to fill this flake with one or more liquids by one or other of its ends. 2 or 3.

FIG. 4 represents a straw where the first plugging means comprises a tripartite plug 4, and where a conventional hollow needle 18 has been installed, by perforation of the second plug ECA 9, at the factory. A bicone "double male Luer "connects this conventional hollow needle to another conventional hollow needle. The beveled tip of the needle 18 protrudes inside the tube the plug of a certain length. This overshoot is provided in the event of injection of liquid by the needle 18 to obtain the creation of a gas bubble whose length will be all the more important as the tip of the needle 18 advances inside. of the tube. Indeed, during a loading of the tube by a liquid, the aspiration by the end 2 of the tube creates a kind of piston of liquid which goes in magnification and which moves towards the tripartite stopper until reaching the powder of the segment 5, which stops the suction by clogging. There thus remains a gas bubble situated between the bevelled tip of the needle and the stopper 9. The gas is normally air, but may be another inert gas if the tube has been filled with another gas.

FIG. 5 represents a straw where the first plugging means comprises an ECA plug 19 as well as a tripartite plug 4 and the second plugging means an ECA plug 9 equipped with a needle 15 with male end, adapted to be connected to a female Luer cone of a conventional injection needle. The tripartite plug 4 was moved during manufacture towards the inside of the tube to make room for the plug 19. A space 21 filled with gas, in particular air, exists between the plug 19 and the tripartite plug 4. This space is provided for allow to open the beveled point of a needle having passed right through the plug 19. This space 21 is not, however, in general essential.

FIG. 6 represents an embodiment of the straw of FIG. 5 in which a conventional needle 22 which passes through the plug 19 and a needle 23 with male end in truncated cone without guard which have been installed in the factory crosses the plug 9.

FIG. 7 schematically represents an apparatus intended for the automatic positioning of the hollow needle with male end 15 or 23 in a straw according to the invention with guidance along the axis of the tube 1.

The apparatus consists of a part 24 which holds the tube 1 in a gutter and a part 25 which holds the needle 15 along the axis of the tube. A translation of 25 approaching 24 has pushed the needle 15 into the desired position.

All of these previous straws are placed for sale under sterile conditions in individual sealed packages in a sterile atmosphere.

A flake fitted as in FIG. 5 with an elastomeric plug 19 not equipped with a needle and an ECA plug 9 not equipped with a needle as in FIG. 3 allows the manufacture in the factory of a flake which contains between its two ECA caps 9, 19 an inert atmosphere which can be chosen in particular from air, carbon dioxide, nitrogen, rare gases, or even a culture medium confined between two gas bubbles.

The general operation of the flakes according to the invention will now be described.

The straw like that of FIG. 3 does not have a three-part stopper 4 or 104, it is possible to inject or remove any fluid through one or other of the ends 2, 3 of the tube 1 provided that the two ECA 9 stoppers are each pierced by a hollow needle.

The tearing of the needles from their ECA cap recreates a seal. The needles are for single use and discarded after use under conditions defined according to the risks of contamination and toxicity.

Thus the flakes according to the invention allow the storage in flakes of doses of any substance which is not chemically aggressive for the components of the tube, in initially liquid, gaseous, or particulate form. The particles must be fine enough to be able to flow through the channel of a needle.

These flakes are perfectly suited for cryogenic storage and conversation of biological liquids, cells, animal or plant gametes, microbes, viruses.

As in the flakes of the prior art, it is possible to collect the content of the flake by cutting the tube by a cutting tool perpendicular to the axis in an area of the adjacent tube, but not opposite. cap screw closing the end 3 of the tube. But for toxic or dangerous products it is preferable to cut the tube and the ECA 9 stopper in elastomer simultaneously so as to leave a rest of the stopper in the form of a more or less thin seal.

These new flakes allow the handling and storage of small doses of chemically or biologically dangerous products under very improved protection conditions.

Conversely, these new flakes allow the handling and preservation of various substances, in particular biological substances under improved asepsis conditions.

Like the flakes of the prior art, the flakes according to the invention withstand sudden transitions in both directions between -196 ° C and about 34 ° C. All the components of the flakes are also chosen to be able to withstand sterilization, in particular by irradiation.

As the flakes are advantageously made, in a manner already known, in a crystal quality material with a wall thickness of approximately 10/100 mm, their transparency and optical quality is such that it is possible to look with a binocular magnifier. passes inside the chaff, especially in the case of gametes, to observe the early stages of embryo development.

Example of fertilization of human gametes using the straw of Figure 3 :

We take a straw, in styrene butadiene or in methyl methacrylate of crystal quality, with a length of 133 mm and an internal diameter of 2 mm, closed at its two ends by ECA plugs, and which has been previously sterilized by gamma irradiation.

One of the plugs, namely A, is pierced by a bevelled hollow needle which serves as an exhaust orifice, the point of the needle barely emerging from the plug.

The other stopper, namely B, is pierced by a needle mounted at the end of a syringe and filled without air with a culture medium liquid, such as, for example, medium B₂ from Menezo.

The entire interior of the tube is then filled with pink Menezo liquid, without the presence of a gaseous phase, since the tip of the needle for injecting this medium hardly comes out of the stopper B.

The injection needle is removed from the stopper B.

Using another needle at the end of another syringe filled without air with a diluting medium, for example F₁ or Tris medium containing spermatozoa, the cap B is again pricked, this time passing the needle d about 15 mm, and a segment of the diluent medium is injected about 10 mm in length; this causes the outlet needle of the stopper A to leave a corresponding volume in the middle of Menezo.

All needles are removed.

The plug B is then re-drilled using a bevelled hollow needle whose tip barely exceeds the plug.

With a needle and a small capacity syringe, a few drops of a diluent containing one or more oocytes are injected through the stopper A. Excess liquid escapes through the hollow needle of stopper B. This introduction of oocyte (s) into the tube can be checked by examination with a binocular microscope.

All needles are removed.

The tube thus filled is kept horizontal at a temperature in the region of 37 ° C, and the cleavage of the oocytes is checked by visual examination. When the embryos reach the stage of division 4 or 8, they are collected in a Petri dish by cutting the tube by a cutting tool perpendicular to the axis of the tube, that is to say in an area located outside the neighboring stopper embryos, either in an area opposite the stopper.

Each embryo collected can be directly implanted in a recipient woman, or frozen for conservation in a French straw in a medium containing a cryoprotector, such as glycerol, ethylene glycol or propylene glycol. To do this, the embryo can be transferred into a straw, according to the present invention, using removable hollow needles. The embryo is placed in its nourishing medium containing the cryoprotective and it is arranged, by successive injections, so that the embryo is found inside the tube in its medium in the form of a segment of liquid surrounded by an air bubble on each side. Air bubbles act as shock absorbers during volume changes caused by liquid-solid and solid-liquid phase transitions during freezing and thawing. In addition, these air bubbles serve as a separator between different segments of different liquid which one does not wish to mix at the start.

It is thus possible to provide, next to the feeder medium containing the embryo and the cryoprotector, another liquid medium known as defrosting, these two media being in the tube separated by an air bubble. The straw is then frozen for preservation. When thawing, the various media ironing in the liquid state can be mixed by shaking the straw so as to bring the air bubbles to one end of the straw, the thawing medium having its effect on the embryo before transfer of it.

Claims (5)

1. A storage tube for the storage and handling of small quantities of biological substances, which is composed of a rectilinear segment (8) of tube (1) of transparent polymer material comprising at its two ends (2, 3) first and second terminal regions provided with respective sealing plugging means (9, 19), characterised in that said plugging means are each formed by a plug of self-sealing compact elastomer of type ECA which is capable of sealingly adhering to the internal wall of the tube (1) and allowing a hollow needle to pass therethrough, the sealing effect being maintained both around the needle when in position and in the plug after withdrawal of the needle.

2. A storage tube according to claim 1 characterised in that the self-sealing elastomer is selected from thermoplastic rubbers and silicone elastomers which are vulcanisable at ambient temperature.

3. A storage tube according to one of claims 1 and 2 characterised in that the self-sealing elastomer has a Shore hardness of between 20 and 50.

4. Use of a storage tube according to any one of claims 1 to 3 in a biomimetic fertilisation process wherein formed in the tube segment (8) is a plurality of contiguous liquid segments which are capable of forming on respective sides of an intermediate connecting region a charging region of male gametes in the proximity of said first terminal region and a charging region of female gametes in the proximity of the second terminal region.

5. Use according to claim 4 characterised in that the charging region of male gametes is separated from the first plug by a segment of liquid of the same nature as the connecting region while the charging region of female gametes is contiguous with the second plug.

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