WO2011104681A1

WO2011104681A1 - Method for obtaining an implantable anterior and/or posterior corneal lamella from a harvested cornea intended for transplantation and liquid to be used in the method

Info

Publication number: WO2011104681A1
Application number: PCT/IB2011/050771
Authority: WO
Inventors: Mauro Beccaro; Enrico Bettini; Paolo Signori
Original assignee: Bbs S.R.L.
Priority date: 2010-02-25
Filing date: 2011-02-24
Publication date: 2011-09-01
Also published as: ITVR20100037A1; IT1398592B1

Abstract

A method for obtaining an implantable anterior and/or posterior corneal lamella from a harvested cornea intended for transplantation comprises the operating steps of taking a harvested cornea intended for transplantation, evaluating its initial thickness, inserting it in a liquid with a hyper- deturgescing action, compared with a preserving liquid, and keeping it in said liquid until it thickness has been reduced by a predetermined level, removing the cornea from the hyper-deturgescing liquid and cutting it to separate the anterior part and the posterior part of the cornea at the stroma or the Descemet's membrane to obtain respectively the anterior lamella and the posterior lamella. Also claimed is a hyper-deturgescing liquid, compared with a liquid for preserving corneas, to be used in a method for obtaining an implantable anterior and/or posterior corneal lamella from a harvested cornea intended for transplantation which was previously preserved in a preserving liquid, for causing deturgescence of the harvested cornea.

Description

METHOD FOR OBTAINING AN IMPLANTABLE ANTERIOR AND/OR POSTERIOR CORNEAL LAMELLA FROM A HARVESTED CORNEA INTENDED FOR TRANSPLANTATION AND LIQUID TO BE USED IN THE METHOD

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DESCRIPTION

This invention relates to a method for obtaining an anterior and/or posterior corneal lamella which can be implanted in a patient from a harvested cornea intended for transplantation, and a liquid to be used in such a method. This invention does not relate to methods which do not comprise obtaining lamellae which meet the requirements for implanting.

In the cornea transplant sector, in recent years the lamellar keratoplasty technique has become increasingly widespread, no longer involving substitution of the entire cornea with the one harvested from a donor, but instead only involving substitution of the diseased layers of the cornea. Many diseases of the cornea are characterised by the fact that they only affect particular layers of the cornea, without damaging the others. In particular, most diseases only affect one of the layers of the cornea (for example, stroma or endothelium), leaving the other layers healthy.

Therefore, in accordance with the lamellar keratoplasty technique, two cases are possible: substitution of the anterior part of the cornea (for example, due to stroma pathologies) or substitution of the posterior part of the cornea (for example, due to endothelium pathologies).

In the former case the technique involves removing from the patient (normally using a laser or microkeratome) the central zone of the stroma and of the Bowman's membrane and of the epithelium which cover it, and substituting them with a similar insert with or without the epithelium (anterior lamella) obtained from the cornea of the donor. Moreover, usually the stroma is not completely removed, to avoid the risks of accidental damage to the Descemet's membrane below. In contrast, in the latter case substitution of the endothelium is usually performed by removing (by scraping) the endothelium together with the Descemet's membrane and, if necessary, with part of the stroma, and substituting it all with a similar insert (posterior lamella) obtained from the cornea of the donor.

However, over the years, the problem which arose was succeeding in obtaining good anterior and posterior lamellae, that is to say, lamellae which are not oedematous, which have the correct thickness, and, in the case of posterior lamellae, which have a large number of live cells (after implanting, the number of live cells is always significantly reduced). To understand the intrinsic difficulty involved in this, it is important to remember that a preserved harvested cornea normally has an overall thickness of between 500 and 700 pm and that the endothelium and Descemet's membrane each have a thickness of only several tenths of a pm.

The first technique developed for this purpose was a manual technique comprising obtaining the posterior lamellae by detachment of the stroma from the Descemet's with a mechanical pulling action. The main disadvantage of this method based on traumatic manipulation of the cornea is the low percentage success rate linked to the high risk of excessive cell damage, especially of the endothelium.

Alternatively, stroma detachment from the Descemet's membrane can be achieved by injecting an air bubble between them. That technique is described for example in patent WO 2008/155748.

Two more automated techniques were subsequently developed, in which separation of the two lamellae is performed by means of a cut made with a blade or a laser, therefore substantially irrespective of the skill of the surgeon.

For both techniques the cornea to be divided is positioned on an artificial chamber into which liquid or gas is injected to create a pressure similar to the physiological pressure of the eye. In the former case, the cut is made using a microkeratome which is associated with the artificial chamber and whose blade can move either linearly by means of a slide, or with a circular motion. In the latter case, the cut is made using a laser beam which is focused at a predetermined depth in the stroma.

However, even these two automated techniques are not without disadvantages.

The first disadvantage is linked to the fact that each harvested cornea is different both due to physiological characteristics and preservation methods. After being harvested, up until the moment the cut is made, corneas are preserved immersed in preserving liquids which must keep their cells alive and minimise any structural modifications to them.

Therefore, all prior art preserving liquids have ingredients which include one or more osmotic/deturgescing agents, that is to say, substances able to give the liquid an osmotic pressure (normally of the colloid osmotic type, that is to say, an oncotic pressure) which is approximately equal to that of an in vivo cornea, so as to prevent it from swelling excessively due to a build up of water. However, in practice, with all prior art preserving liquids, during preservation there is always a certain amount of swelling (turgescence) of the preserved cornea due to absorption of water from the preserving liquid. A detailed study of such problems is found, for example, in the article "Storage of Human Corneas in Dextran and Chondroitin Sulfate-Based Corneal Storage Medium", Jablonski-Stiemke, Edelhauser, ARCH OPHTALMOL, Vol. 116, May 1998, pp. 627-632.

The introduction to the above-mentioned patent WO 2008/155748 also refers to a technique developed in the 1970s which involves, before implanting a preserved cornea, its insertion in a 4% - 6% high molecular weight (500,000 Da) dextran solution for around twenty four hours, in order to bring down the swelling and return it to a thickness similar to the original thickness. However, it should be noticed that in reality the solution referred to in said patent is none other than a preserving liquid, although with greater deturgescing properties than the culture medium which was used at the time for preserving corneas for a period of up to four weeks. Said circumstance is demonstrated in the more recent patent application US 2006/228639 relating to preserving liquids and which describes as an example embodiment the use of dextran with a molecular weight of at least 500,000 Da in concentrations even of up to 10% by weight. All such liquids are therefore deturgescing preserving liquids.

In addition to the preserving liquids there are some prior art liquids which can reduce swelling of the cornea but at the same time do not allow the obtainment of lamellae implantable in a patient, but only portions of cornea for use in research. One example of such liquids and of their effect is described in the article "Corneal Preparation of Eye Bank Eyes for Experimental Surgery", Hamaoui et al., Cornea 20(3): 317-20, 2001.

Therefore, returning to the technique for obtaining lamellae, at the moment the cut is made, the cornea to be transplanted is always relatively swollen with significant disadvantages in terms of the results which can be achieved. That is confirmed by the fact that, after implanting, at present a reduction in the thickness of what was implanted (whole cornea or lamella) can always be recorded.

In the case of a cut made with a blade the main problem is the thickness of the posterior lamellae obtained. Said thickness is determined starting with the overall thickness of the cornea, minus the thickness of the minimum anterior lamella obtainable with the blade of the microkeratome (the blade is usually around 300 or 350 pm thick and allows an anterior lamella measuring approximately 350 - 400 μητι to be obtained).

Therefore, at present, since a preserved (and so swollen) cornea has a thickness of around 550 - 700 pm, it is possible to obtain anterior lamellae measuring approximately 400 pm and posterior lamellae measuring 200 - 300 pm, much more than the few tenths of a μητι that ideally one would aim to obtain.

Moreover, preserved corneas, being swollen, have an accentuated curvature which hinders correct cutting, because the microkeratome only allows the cut to be made in one plane and because that plane must be completely within the thickness of the stroma to avoid damaging the cellular layers. Consequently, if there is excessive convexity, it may be impossible to make an acceptable cut, although this can normally only be verified after the cut has been made.

Laser cutting also involves problems. In this case, the main problem is the excessive thickness and excessive convexity of corneas which are swollen with water.

With the laser cutting devices currently known, on one hand there is an inverse limitation between the diameter and depth of the cut (an increase in one reduces the other), and on the other hand the power of the beam is increasingly attenuated as it penetrates the cornea. Consequently, if the cornea is very thick, the laser cannot operate deep enough to make the required cuts. Secondly, when the cornea is swollen, the collagen fibres which make up the stroma are spaced out due to the presence of water, making it difficult, if not impossible, to focus the laser beam on them. Therefore, in most cases the cut surface is irregular, leaving the patient with a sight deformation.

Moreover, lamellae obtained using the prior art methods have the disadvantage of being too flaccid, which may easily result in their deterioration during subsequent handling during the implanting step.

The consequence of the above is that, at present, many surgeons choose not to use harvested corneas which are too thick, due to the impossibility of obtaining posterior lamellae which are transplantable and have suitable thickness.

It should be remembered that a swollen (oedematous) cornea is not transparent, meaning that after the transplant several weeks may have to go by before the patient can begin to see well again, that is to say, before the physiological functions of the eye succeed in using osmosis to remove the excess water. It is obvious that such a convalescence period is longer the greater the amount of water there is in the transplanted tissue.

Unlike what was just described, it should also be noticed how, in the case of the technique for producing lamellae described in patent WO 2008/155748, which involves creating an air cushion between the stroma and the Descemet's membrane, application of the method is much simpler if the cornea is swollen. Injection of the air bubble is much easier in corneas with increased thickness and reduced rigidity. Therefore, it is no coincidence that said patent does not include the use of deturgescing liquids with the method for producing lamellae.

Finally, it should be noticed that, until now, all prior art deturgescing liquids intended for use for the preservation and/or treatment of harvested corneas intended for transplantation have had a combination of ingredients such that a cornea immersed in them can remain viable without an excessive increase in thickness or, in some cases, with a reduction in thickness. However, all such deturgescing liquids share the feature of being able to extract water from the cornea only until the quantity of water reaches a safety level which is higher than the physiological water content of a cornea. Examples of such deturgescing liquids able to keep the water content (and therefore the thickness) of the cornea at a value which is not too much above the physiological value are both the de-swelling liquid described in the introduction to patent WO 2008/155748 and the liquid which is the subject matter of patent US 2006/228693 (although in this case only uses with cow or pig corneas are described, which have significant differences compared with human corneas both relative to the thickness and the kinetics in contact with a deturgescing liquid).

In this context, the technical purpose which forms the basis of this invention is to provide a method for obtaining an implantable anterior and/or posterior corneal lamella from a harvested cornea intended for transplantation, and a liquid to be used in the method, which overcome the mentioned disadvantages.

In particular, the technical purpose of this invention is to provide a method for obtaining an implantable anterior and/or posterior corneal lamella from a harvested cornea intended for transplantation, and a liquid to be used in the method, which allow the precise and repeatable obtainment of lamellae whose thickness is decidedly less than those obtainable with the prior art methods.

Another technical purpose of this invention is to provide a method for obtaining an implantable anterior and/or posterior corneal lamella from a harvested cornea intended for transplantation which allows the obtainment of lamellae which are more transparent than those currently obtainable, as well as lamellae which are relatively more rigid.

Yet another technical purpose of this invention is to provide a method for obtaining an implantable anterior and/or posterior corneal lamella from a harvested cornea intended for transplantation, and a liquid to be used in the method, which always allow laser cutting at the desired depths and with cut surfaces which are more regular than in the prior art.

The technical purpose and the aims indicated are substantially achieved by a method for obtaining an implantable anterior and/or posterior corneal lamella from a harvested cornea intended for transplantation and by a liquid to be used in the method as described in the appended claims.

Further features and the advantages of this invention are more apparent in the detailed description which follows of several preferred, non-limiting embodiments of a method for obtaining an implantable anterior and/or posterior corneal lamella from a harvested cornea intended for transplantation and the liquid to be used in the method.

Therefore, hereinafter any reference to lamellae and corneas shall be understood to refer respectively to lamellae which meet the requirements for implanting in a patient and to corneas intended for transplantation and preserved for that purpose.

As indicated, the method according to this invention allows an anterior and/or posterior corneal lamella to be obtained from a healthy harvested cornea. In general, starting with a healthy harvested cornea, the method always allows two lamellae to be obtained, even if the thickness can only be checked on one lamella at a time because there is only one cut surface. Obviously, the first step of the method is to take a harvested cornea. This is usually a harvested cornea preserved until that moment in a conventional preserving liquid. Consequently, at the start of the method according to this invention, the cornea is swollen and therefore its thickness and convexity are greater than in the physiological conditions.

In the preferred embodiment the method comprises a step of evaluating the initial thickness of the cornea. Said step may advantageously be carried out either when the cornea is still immersed in the preserving liquid, or after removing it from the preserving liquid and inserting it in a hyper- deturgescing liquid.

It should be noticed that in the context of this invention the terms hyperosmotic, hyper-deturgescing, hyper-oncotic will be used without distinction as synonyms to indicate a liquid whose osmotic pressure is greater than that of any liquid suitable for preserving corneas (corneal storage medium or corneal culture medium, depending whether it is intended for preserving the corneas respectively at around 4°C or at around 31 °C). Therefore, it is a liquid able to extract water from a cornea that was previously stored in any preserving liquid. Thus, the liquid according to this invention, in itself, cannot be considered a preserving liquid because, in it, a cornea can only survive for a limited time. If left in it too long, the cornea gradually loses its water content until the endothelial cells die.

In other words, in the preferred embodiment, the hyper-deturgescing liquid is able to reduce the quantity of water present in the cornea even below the quantity which is physiologically present in corneas in vivo. For a more detailed description of the hyper-deturgescing liquid and for some examples of its combination of ingredients, refer to the description below. Finally, it should be noticed that, since in the preferred embodiment the liquid is of the colloid type, for the purposes of this invention reference is made without distinction to its osmotic pressure or its oncotic pressure.

One of the main advantages of the liquid is that it cannot damage corneas at least for contact times of several hours (advantageously many), so that the deturgescing process is completely reversible without risks for the possibility of implanting the lamellae obtained. In other words, after a cornea has been immersed in the hyper-deturgescing liquid for several hours (and so its swelling has gone down), it can be removed, immersed in a preserving liquid again and reacquire the initial water content without any damage to its various parts.

In accordance with this invention, the cornea is therefore immersed in the hyper-deturgescing liquid until its thickness has been reduced at least by a predetermined level and the cornea still meets the requirements for implanting.

The predetermined level may be established in advance for all corneas (in absolute or percentage terms), or on each occasion based on the initial thickness measured. For example, tests carried out by the Applicant demonstrated that good results can be obtained when during the step in which the cornea is kept in the hyper-deturgescing liquid the thickness of the cornea is reduced by at least around 100 μιη irrespective of the initial thickness.

As indicated, the evaluation of the initial thickness may be performed either before inserting the cornea in the hyper-deturgescing liquid, or immediately afterwards.

Advantageously, in both cases the evaluation of the initial thickness of the cornea may be performed by examining the cornea while it is inserted in a container and immersed in the preserving bath/in the hyper-deturgescing liquid.

Since the time needed to achieve the predetermined reduction in thickness cannot usually be precisely forecast in advance because it depends on the physiology and the metabolism of the cornea (advantageously, it will be approximately several hours), in the preferred embodiment the step of evaluating the thickness of the cornea is repeated several times while it is kept in the hyper-deturgescing liquid, to check the gradual reduction of its swelling from the initial thickness to the predetermined one.

Again in this case, the evaluation of the thickness of the cornea is carried out by examining the cornea inserted in a container and immersed in the hyper-deturgescing liquid.

All of the evaluations of the thickness of the cornea described above may be carried out using any suitable technology, such as OCT (optical coherence tomography) systems, ultrasound pachymetry, and confocal and endothelial microscopes.

Once the desired thickness has been reached, the method comprises removing the cornea from the hyper-deturgescing liquid and cutting to separate the anterior part and the posterior part of the cornea (at the stroma but, preferably as close as possible to the Descemet's membrane) to obtain respectively the anterior lamella and the posterior lamella. Any technique, conventional or not, may be used to separate the two parts by cutting.

It should also be noticed that, advantageously, the predetermined thickness may be selected in such a way that at the moment when the cornea is removed from the hyper-deturgescing liquid to proceed with cutting, it is relatively flat and rigid compared with its initial condition (that is to say, its condition in the preserving liquid). Thanks to the partial dehydration to which it was subjected and which reduced its swelling, the cornea is less convex than it was at the start (and in some cases even than when it was in vivo) and it is more rigid thanks to the reduced water content in the fibrous structures.

As regards the step of cutting to separate the anterior part of the cornea from the posterior part, as indicated it may be performed in any way, but at present it is preferably carried out using a cut with a blade or a cut with a laser.

Thanks to the fact that the cornea is less thick and less concave than with prior art methods, on one hand a cut with a blade (still with the same thickness - for example 350 - 400 pm) allows the obtainment of at least one lamella which is much thinner than with the prior art methods (for example with treated cornea thicknesses of around 450 - 600 pm, therefore with a reduction of around 50 - 100 pm compared with its initial value, it is possible to obtain one of the two lamellae, advantageously the posterior one, having a thickness of 100 - 200 pm or even less). On the other hand, a cut with a laser allows, the diameter being equal, incisions which are relatively deeper relative to the cornea (the absolute cutting depth being equal, a reduced thickness corresponds to relatively greater penetration) and cuts which are cleaner and more precise (in corneas whose swelling has gone down the collagen fibres are closer together and it is easier to focus the laser on them).

A clear result of what is described above is that one of the main aspects of this invention is that it provides a hyper-deturgescing liquid for reducing the thickness of the cornea below its physiological value.

Therefore, the hyper-deturgescing liquid is also part of the subject matter of this invention. In general terms it comprises at least water and osmotic agents (the term osmotic agents refers to those substances able to significantly increase the osmotic pressure of the liquid) in quantities such that they give it a hyper-osmotic action compared with a liquid suitable for preserving corneas, so that it can cause deturgescence of the harvested cornea even to below its physiological level. In any case, the liquid is composed in such a way that it does not damage the cornea at least for immersion times of several hours.

In general, any deturgescing, osmotic or oncotic (colloid or not) agent may be used to make the hyper-deturgescing liquid according to this invention. For example, the following osmotic, oncotic, deturgescing agents may be used:

- glycosaminoglycans (sulfated and not);

- proteins;

- anionic and cationic polysaccharides;

- natural and synthetic hydrophilic and hydrophobic colloids;

- non-glycosaminoglycans;

- glycerol;

- urea;

- mannitol;

- polyethylene glycol and propylene glycol copolymers.

Moreover, in a preferred embodiment, the liquid comprises two or more different osmotic agents. In particular, the liquid advantageously comprises both first osmotic agents having a molecular weight such that they cannot enter the layers of the cornea, and second osmotic agents having a molecular weight such that they can fit between the layers of the cornea. Advantageously, both the first osmotic agents and the second osmotic agents are present in a quantity of between 3% and 8% by weight, preferably between 4% and 6%. In the preferred embodiments, the first osmotic agents and the second osmotic agents are both present in a quantity equal to around 5% by weight.

In a preferred embodiment, in particular, the liquid comprises dextran with a molecular weight equal to 500,000 Daltons (as dextran T500) as the first osmotic agent and dextran with a molecular weight equal to 40,000 Daltons (as dextran T40) as the second osmotic agent. Moreover, the liquid preferably comprises first osmotic agents and second osmotic agents in a proportion relative to each other, by weight, of between 0.5 and 2, and preferably between 0.8 and 1.2.

In the most common embodiments, the liquid also comprises one or more antioxidants, antibiotics, culture medium (that is to say, a substance able to guarantee nourishment for the cells of the cornea such as MEM - Minimum Essential Medium), vitamins, growth factors, serums, products derived from humans and buffer systems.

In a preferred composition, the hyper-deturgescing liquid has a purified water base and comprises, by weight, at least:

b-MERCAPTOETHANOL 25 - 35 ppm

DEXTRAN with molecular weight 500 kDa 4.3 - 4.7 %

DEXTRAN with molecular weight 40 kDa 4.3 - 4.7 %

GENTAMICIN SULFATE 100 - 150 ppm

MEM (powdered) 1.1 - 1.5 %

SODIUM PYRUVATE 80 - 120 ppm

SODIUM BICARBONATE 0.1 - 0.3 %.

A specific example of the composition of the hyper-deturgescing liquid provided by the Applicant is:

PURIFIED WATER 20 ml

b-MERCAPTOETHANOL 0.0007 ml

DEXTRAN with molecular weight 500 kDa 1.0 g

DEXTRAN with molecular weight 40 kDa 1.0 g

GENTAMICIN SULFATE 0.00286 g

MEM (powdered) 0.2996 g

SODIUM PYRUVATE 0.0022 g

SODIUM BICARBONATE 0.044 g

In accordance with what is described above, said liquid may remain in contact with corneas without damaging them even for many hours (even for an entire day), and when the immersion ends the lamellae which can be obtained are definitely implantable in a patient.

Finally, this invention also relates to a hyper-deturgescing liquid (compared with a liquid for preserving corneas) to be used in a method for obtaining an anterior and/or posterior corneal lamella from a harvested cornea, for causing deturgescence of the harvested cornea which was previously preserved in a preserving liquid (deturgescence which may also go beyond the physiological level of the cornea), as well as the use of first osmotic agents having a molecular weight such that they cannot enter the layers of a harvested cornea and second osmotic agents having a molecular weight such that they can fit between the layers of the harvested cornea for making a liquid that has a hyper-deturgescing action, compared with a preserving liquid, to be used in a method for obtaining an anterior and/or posterior corneal lamella from a harvested cornea.

This invention brings important advantages.

First, thanks to this invention not only is it possible to obtain lamellae with a thickness which is less than those obtainable with the prior art methods, but it is also possible to do so in a precise and repeatable way. With the liquid according to this invention it is possible to decide in advance the thickness to which the cornea will be brought before it is cut.

Moreover, thanks to this invention it is always possible to cut the cornea using a laser or a blade to the desired depths. The depth of the cut is directly linked to the thickness of the cornea at the moment the cut is made. Second, thanks to this invention it is possible to obtain lamellae which are less oedematous (and therefore more transparent) than those currently obtainable, thus providing a great advantage in terms of the time needed for recovery of sight by the patient in which they will be implanted.

Moreover, thanks to this invention it is possible to obtain transplantable posterior lamellae from harvested corneas intended for transplantation having any thickness.

Finally, it should be noticed that this invention is easy to produce and that even the cost linked to implementing the invention is not very high.

The invention described above may be modified and adapted in several ways without thereby departing from the scope of the inventive concept. Moreover, all details of the invention may be substituted with other technically equivalent elements and the materials used, as well as the shapes and dimensions of the various components, may vary according to requirements.

Claims

1. A method for obtaining an implantable anterior and/or posterior corneal lamella from a harvested cornea intended for transplantation, characterised in that it comprises the operating steps of:

taking a harvested cornea intended for transplantation which was previously preserved in a preserving liquid;

evaluating the initial thickness of the cornea;

inserting the cornea in a hyper-deturgescing liquid, that is to say, a liquid having an osmotic pressure which is greater than that of a liquid suitable for preserving the cornea;

identifying a minimum reduction in thickness to which the cornea must be subjected;

keeping the cornea immersed in the hyper-deturgescing liquid until its thickness has undergone at least the predetermined reduction;

removing the cornea from the hyper-deturgescing liquid; and

separating the anterior part and the posterior part of the cornea at the stroma or the Descemet's membrane by cutting;

the anterior and/or posterior lamella intended for transplantation respectively corresponding to the anterior part and/or the posterior part of the cornea obtained in this way.

2. The method according to claim 1 , characterised in that it also comprises repeating the step of evaluating the thickness of the cornea while it is being kept in the hyper-deturgescing liquid, for checking the gradual reduction of its thickness until it reaches at least the predetermined reduction.

3. The method according to either of the foregoing claims, characterised in that the step of cutting to separate the anterior part of the cornea from the posterior part is carried out by means of a cut with a blade or a cut with a laser.

4. The method according to any of the foregoing claims, characterised in that the step of evaluating the initial thickness of the cornea may be carried out before inserting the cornea in the hyper-deturgescing liquid or at the start of the step of keeping the cornea in said liquid.

5. The method according to claim 4, characterised in that the step of evaluating the initial thickness of the cornea is carried out before inserting the cornea in the hyper-deturgescing liquid and also being characterised in that the step of evaluating the initial thickness of the cornea is carried out by examining the cornea inserted in a container and immersed in a preserving bath.

6. The method according to claims 2 and 5, characterised in that the step of evaluating the thickness of the cornea during the step of keeping it in the hyper-deturgescing liquid is carried out by examining the cornea inserted in a container and immersed in the hyper-deturgescing liquid.

7. The method according to claim 4, characterised in that the step of evaluating the initial thickness of the cornea is carried out at the start of the step of keeping it in the hyper-deturgescing liquid and also being characterised in that the step of evaluating the thickness of the cornea is carried out by examining the cornea inserted in a container and immersed in the hyper-deturgescing liquid.

8. The method according to any of the foregoing claims, characterised in that the step of keeping the cornea in the hyper-deturgescing liquid lasts at least until the thickness of the cornea is reduced by at least approximately 50 - 10Ό μηι.

9. The method according to any of the foregoing claims, characterised in that the step of inserting the cornea in a hyper-deturgescing liquid comprises the use of a liquid comprising both first osmotic agents having a molecular weight such that they cannot enter the layers of the cornea, and second osmotic agents having a molecular weight such that they can fit between the layers of the cornea.

10. The method according to claim 9, characterised in that it uses a hyper- deturgescing liquid comprising dextran with a molecular weight of 500 kDa as a first osmotic agent and dextran with a molecular weight of 40 kDa as a second osmotic agent.

11. The method according to claim 9 or 10, characterised in that it uses a hyper-deturgescing liquid comprising first osmotic agents and second osmotic agents in a proportion to each other by weight of between 0.5 and 2, and preferably between 0.8 and 1.2 and/or in a quantity of between 3% and 8% by weight and preferably between 4% and 6%.

12. A liquid to be used in a method for obtaining an implantable anterior and/or posterior corneal lamella from a harvested cornea intended for transplantation, characterised in that it is liquid with a hyper-deturgescing action, compared with a liquid suitable for preserving a cornea, thus being able to cause deturgescence, even to below its physiological level, of a harvested cornea intended for transplantation which was previously preserved in a preserving liquid.

13. The liquid according to claim 12, characterised in that it comprises both first osmotic agents having a molecular weight such that they cannot enter the layers of the cornea, and second osmotic agents having a molecular weight such that they can fit between the layers of the cornea.

14. The liquid according to claim 13, characterised in that it comprises dextran with a molecular weight of 500 kDa as a first osmotic agent and dextran with a molecular weight of 40 kDa as a second osmotic agent.

15. The liquid according to claim 13 or 14, characterised in that it comprises first osmotic agents and second osmotic agents in a proportion relative to each other, by weight, which is between 0.5 and 2, preferably being between 0.8 and 1.2.

16. The liquid according to claim 3, 14 or 15, characterised in that both the first osmotic agents and the second osmotic agents are present in a quantity of between 3% and 8% by weight, preferably between 4% and 6%.

17. The liquid according to any of the claims from 12 to 16, characterised in that it also comprises one or more of the following: antioxidants, antibiotics, culture medium, vitamins, growth factors, serums, products derived from humans and buffer systems.

18. The liquid according to any of the claims from 12 to 17, characterised in that it has a purified water base and comprises, by weight, at least:

b-mercaptoethanol 25 - 35 ppm

dextran with molecular weight 500 kDa 4.3 - 4.7 %

dextran with molecular weight 40 kDa 4.3 - 4.7 %

gentamicin sulfate 100 - 150 ppm

MEM (powdered) 1.1 - 1.5 %

sodium pyruvate 80 - 120 ppm

sodium bicarbonate 0.1 - 0.3 %.

19. Use of first osmotic agents having a molecular weight such that they cannot enter the layers of a harvested cornea and second osmotic agents having a molecular weight such that they can fit between the layers of a harvested cornea for producing a hyper-deturgescing liquid to be used in a method for obtaining an implantable anterior and/or posterior corneal lamella from a harvested cornea intended for transplantation.

20. The use according to claim 19, characterised in that it uses dextran with a molecular weight of 500 kDa as a first osmotic agent and dextran with a molecular weight of 40 kDa as a second osmotic agent.

21. The liquid according to claim 9 or 20, characterised in that it uses first osmotic agents and second osmotic agents in a proportion relative to each other, by weight, which is between 0.5 and 2, preferably being between 0.8 and 1.2.

22. The liquid according to claim 19, 20 or 21 , characterised in that it uses both first osmotic agents and second osmotic agents in a quantity of between 3% and 8% by weight, preferably between 4% and 6%.