PRESERVATION AND TRANSPORT OF AN EX VIVO BIOLOGICAL SAMPLE COMPRISING ULTRASOUND APPLICATION

Abstract

The present invention relates to a device for transport and preservation of an ex vivo biological sample and corresponding method. The device (1) comprises a chamber (2) for containing the biological sample (100), delimitated by walls (4) made of a thermal insulating material. The device, furthermore, incorporates cooling means (6) that keep the temperature inside the chamber (2) below the temperature outside the device (1). Finally, an ultrasound system suitable for generating and applying ultrasound on the biological sample (100) is provided. The invention also proposes a method for transport and preservation which combines applying cooling and ultrasound to reduce cell damage in the biological sample.

Claims

1. A device (1) for transport and preservation of an ex vivo biological sample (100) for subsequent transplant in a living human being or animal, comprising a chamber (2) for containing said biological sample (100), delimitated by walls (4) made of a thermal insulating material and cooling means (6) for keeping the temperature inside said chamber (2) below the temperature outside said device (1), characterized in that it further comprises an auxiliary container (14) for containing a preservation solution, without external oxygen delivery and for containing said biological sample (100) immersed in said preservation solution and at least an ultrasound system suitable for generating and applying ultrasound on said biological sample (100).

2. The device (1) according to claim 1, characterized in that said auxiliary container (14) is closable.

3. The device (1) according to claim 1, characterized in that said ultrasound system is suitable for emitting said ultrasound at a frequency comprised between 25 kHz and 1 MHz and a sound intensity comprised between 0.01 and 2 W/cm.sup.2.

4. The device (1) according to claim 3, characterized in that said sound intensity is comprised between 0.02 and 1 W/cm.sup.2.

5. The device (1) according to claim 1, characterized in that said cooling means (6) are suitable for keeping the temperature inside said chamber (2) between 0 and 15° C.

6. The device (1) according to claim 5, characterized in that said cooling means (6) are suitable for keeping the temperature inside said chamber (2) between 2 and 10° C.

7. The device (1) according to claim 1, characterized in that it comprises a sheet-like support (10) in said chamber (2) suitable for supporting said closable auxiliary container (14) containing said biological sample (100), said sheet-like support (10) being able to vibrate freely when said ultrasound is applied, and in that said ultrasound system comprises at least one transducer (8) mounted on at least one of the walls (4) of said sheet-like support (10).

8. The device (1) according to claim 7, characterized in that said at least one transducer (8) is mounted on the face of said sheet-like support (10) opposite the support surface (12) for said biological sample (100) to apply said ultrasound towards said support surface (12).

9. The device (1) according to claim 7, characterized in that said sheet-like support (10) is made of metal.

10. The device (1) according to claim 7, characterized in that sheet-like support (10) is a tray adapted for containing a fluid.

11. (canceled)

12. The device (1) according to claim 1, characterized in that said auxiliary container (14) comprises a false bottom (18) located away from the base of said auxiliary container (14), and said false bottom (18) being in fluid communication with the rest of said auxiliary container (14).

13. A method for transport of an ex vivo biological sample (100) for subsequent transplant in a living human being or animal, characterized in that it comprises the following steps: [a] removing blood from said biological sample (100) under cold conditions and rapidly cooling said biological sample (100), [b] keeping said biological sample (100) immersed in a preservation solution without external oxygen delivery by placing said biological sample in a chamber (2) delimitated by walls (4) made of a thermal insulating material, and [c] irradiating said sample with ultrasound.

14. The method according to claim 13, characterized in that said biological sample (100) is placed immersed in an auxiliary container (14) containing said preservation solution, and said auxiliary container (14) is placed in said chamber (2).

15. The method according to claim 13, characterized in that in said irradiation step for irradiating said biological sample (100), the ultrasound has frequencies comprised between 25 kHz and 1 MHz and a sound intensity comprised between 0.01 and 2 W/cm.sup.2

16. The method according to claim 15, characterized in that said ultrasound has an intensity comprised between 0.02 and 1 W/cm.sup.2.

17. The method according to claim 13, characterized in that it further comprises a cooling step for cooling the temperature in said chamber (2) to a temperature between 0 and 15° C.

18. The method according to claim 17, characterized in that in said cooling step the temperature in said chamber (2) is kept between 2 and 10° C., and particularly preferably between 2 and 6° C.

19. The method according to claim 13, characterized in that it further comprises a step of placing said auxiliary container (14) on a sheet-like support (10) that is a tray adapted for containing a fluid and said tray being able to vibrate freely when said ultrasound is applied.

20. Use of a device (1) for transport and preservation of an ex vivo biological sample (100) according to claim 1, characterized in that said biological sample (100) is maintained immersed in a preservation solution without oxygen delivery under hypothermal conditions and said sample (100) is irradiated with ultrasound such that the viability, functionality and interaction between the different cells of said biological sample (100) are preserved for said biological sample (100) to be used in subsequent laboratory research.

21. The device (1) according to claim 4, characterized in that said sound intensity is comprised between 0.02 and 0.1 W/cm.sup.2.

22. The device (1) according to claim 6, characterized in that said cooling means (6) are suitable for keeping the temperature inside said chamber (2) between 2 and 6° C.

23. The method according to claim 14, characterized in that said auxiliary container (14) is closable.

24. The method according to claim 13, characterized in that in said ultrasound is applied in a pulsed manner.

25. The method according to claim 16, characterized in that said ultrasound has an intensity comprised between 0.02 and 0.1 W/cm.sup.2.

26. The method according to claim 18, characterized in that in said cooling step the temperature in said chamber (2) is kept between 2 and 6° C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Further advantages and features of the invention will become apparent from the following description, in which, without any limiting character, preferred embodiments of the invention are disclosed, with reference to the accompanying drawings in which:

[0044] FIG. 1 shows an exploded perspective view of a first embodiment of a device for transport of an ex vivo biological sample.

[0045] FIG. 2 shows a longitudinally sectioned front view of the device of FIG. 4.

[0046] FIG. 3 shows a top plan view of a first embodiment of the support tray for the biological sample of the device of FIG. 1.

[0047] FIG. 4 shows a side view of the tray of FIG. 3.

[0048] FIG. 5 shows a second embodiment of the support tray for the biological sample.

[0049] FIG. 6 shows a second embodiment of the device for transport according to the invention.

[0050] FIG. 7A shows a third embodiment of the device for transport according to the invention.

[0051] FIG. 7B shows an alternative embodiment of the auxiliary container of the device of FIG. 7A.

[0052] FIGS. 8A to 13 show percentage of protection of biological samples of liver and kidney with respect to conditions in the state of the art.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0053] FIGS. 1 and 2 show a first embodiment of the device 1 for transport and preservation of an ex vivo transplantable biological sample 100 according to the invention. In a particularly preferred manner, the device according to the invention is a portable cooler.

[0054] As has already been previously indicated, the biological sample 100 comprises both organs that can be transplanted between donor and recipient, which can be human or animal, and tissues. Furthermore, the biological sample can also be intended for research without necessarily having to be transplanted.

[0055] The device 1 for transport and preservation according to the invention has a parallelepiped-shaped main isothermal container 16 open on the upper face. The walls 4 made of a thermal insulating material of the main container 16, including its upper lid, delimitate a chamber 2 intended for containing the biological sample 100.

[0056] Cooling means 6 suitable for keeping the temperature inside the chamber 2 below the temperature outside said device 1 are provided inside the main container 16. These cooling means 6 can comprise solutions as different as ice blocks, cooling gel packs or more complex solutions comprising a compressor and a heat exchanger. The device 1 could also consist of a transportable cooler with or without an external power supply. Nevertheless, it is desirable for the solution to be as light as possible in order to not affect transportability of the assembly. The cooling means 6 allow keeping the temperature inside the chamber 2 between 0 and 15° C. In another preferred embodiment, the temperature inside the chamber 2 is kept between 2 and 10° C., and particularly preferably between 2 and 6° C.

[0057] The device 1 comprises in the central part a sheet-like support 10 by way of a metal tray supported inside the chamber 2 such that it can vibrate freely. The tray of this first embodiment can be seen in detail in FIGS. 3 and 4. An aluminum tray with less than 1 mm, and preferably 0.5 mm thick, is used in the embodiment.

[0058] Furthermore, the device comprises an ultrasound system suitable for generating and applying ultrasound on the biological sample 100. To that end, the ultrasound system according to the drawings has an electric signal generator 20, an amplifier 22, a battery 24, and in this case four piezoelectric transducers 8.

[0059] Vibrations are applied to the sheet-like support 10 by way of an aluminum tray through the four transducers 8. Said transducers 8 are mounted on the lower face of the tray. As a result of this configuration, transmission between the transducers and the biological sample 100 is more direct, because vibrations are generated directly below the lower part of the biological sample. In a particularly preferred manner, as can be seen in FIG. 6, the tray can additionally contain water, but to improve transmission of ultrasound, it is envisaged that the biological sample 100 is contained in a bag or container filled with preservation solution. The preservation solutions contemplated herein are, for example, Lactated Ringer's solution, Celsior solution or University of Wisconsin solution.

[0060] In an alternative embodiment shown in FIG. 5, the tray can have transducers 8 on the side walls to generate a lateral vibrational field. The transducers 8 could also alternatively be located both on side walls and on the bottom of the tray.

[0061] The ultrasound transducers 8 transmit mechanical waves having a frequency comprised between 25 kHz and 1 MHz and a sound intensity comprised between 0.1 and 2 W/cm.sup.2 into said chamber 2 during transport of said biological sample 100. The intensity can preferably be comprised between 0.02 and 1 W/cm.sup.2, and still more preferably between 0.02 and 0.1 W/cm.sup.2. The higher the sound intensity used, the further away the transducers 8 will be and/or the larger the amount of preservation fluid or coolant will be placed in the device 1 to meet the objective of preventing local heating in the biological sample 100. Furthermore, according to the invention the ultrasound can be applied continuously. Alternatively, the ultrasound can also be applied intermittently, i.e., it is not applied during the entire time transport lasts. Alternatively, it can also be applied in a pulsed manner and/or at different frequencies, either continuously or intermittently.

[0062] In a particularly preferred manner, the biological sample 100 is contained in a receptacle containing a preservation solution, which improves the results of applying ultrasound.

[0063] Other embodiments of the device 1 for transport according to the invention which share many of the same features described in the preceding paragraphs are shown below. Accordingly, only those elements that are different between such embodiments will be described hereinafter, whereas for common elements reference is made to the description of the first embodiment.

[0064] The embodiment of the device 1 of FIG. 6 differs primarily in that the sheet-like support 10 is integrated directly in the walls 4 of the main container 16.

[0065] Finally, in the embodiment of the device in FIG. 7A, an auxiliary container 14 is provided inside the main container 16 with a preservation solution. In a particularly preferred manner, the auxiliary container 14 is closable, with rigid walls and a lid hinged on one of its walls. In a particularly preferred manner, when the transducers are located in the lower part of the tray, the auxiliary container 14 has dimensions intended for occupying the entire emission surface of the transducers 8. As a result of the auxiliary container 14 being filled with preservation solution, the ultrasound reaches the biological sample 100 in a more efficient manner. In a particularly preferred manner, the auxiliary container 14 is manufactured by thermoforming using a formable plastic, such as for example high density polyethylene, polypropylene, polyethylene terephthalate or the like. Also in a particularly preferred manner, the material will be transparent to enable seeing the biological sample 100 without needing to open the auxiliary container 14.

[0066] FIG. 7A shows an alternative embodiment of the auxiliary container 14. In this case, the auxiliary container 14 comprises a false bottom 18 located away from the base of said auxiliary container 14, and said false bottom 18 being in fluid communication with the rest of said auxiliary container 14. The false bottom thereby places the biological sample 100 away from the support surface of the auxiliary container 14. In this case, the false bottom 18 consists of a sheet of rigid plastic supported on the side walls of the auxiliary container 14. Then to achieve proper fluid communication, a plurality of openings 20 that allow the passage of the preservation solution is provided in this case, such that said solution receives the direct effect of the ultrasound coming from the lower transducers 8.

[0067] Finally, in a particularly preferred manner, the auxiliary container 14 contains a preservation fluid under sterile conditions from the group consisting of Lactated Ringer's preservation solution, Celsior preservation solution or University of Wisconsin preservation solution. This allows a much more hygienic handling of the biological sample under more suitable conditions for the proper preservation thereof.

[0068] The embodiments described up until now represent non-limiting examples, such that the person skilled in the art will understand that beyond the examples that are shown, a number of combinations of the claimed features are possible within the scope of the invention.

EXPERIMENTAL EXAMPLES

[0069] Different tests conducted to put the method according to the invention into practice are described below.

[0070] Methodology

[0071] The experimental protocol was carried out from livers and kidneys of Landrace pigs. The kidney and liver were perfused with preservation solution at 4° C. to remove blood contained in the organ, and the organ was kept under cold conditions, bathed in ice until the organs were immersed in different preservation solutions. Next the biological samples were placed in the cooler and kept in the cooler with or without ultrasound for 8 hours (liver) and 24 hours (kidney). All the procedures were performed under anesthesia and the study respected the European Union regulations concerning experiments using animals (Directive 86/609/EEC).

[0072] Experimental Design

[0073] Protocol 1

[0074] The experimental groups formed were the following: [0075] GROUP 1: cold ischemia group in the conventional transport system and with preservation solution. This group was divided into different subgroups depending on the preservation solution used. [0076] 1.1) Perfusion of liver grafts (n=10) and kidney grafts (n=10) in Lactated Ringer's solution at 4° C. and preservation of such organs with Lactated Ringer's solution in the conventional transport system for 8 and 24 hours for liver and kidney, respectively, between 2-6° C.; [0077] 1.2). The same as 1.1 but using Celsior solution for washing and preserving the organ; [0078] 1.3) The same as 1.1 but using UW (University of Wisconsin) solution for washing and preserving the organ. [0079] GROUP 2: cold ischemia group in the system with ultrasound (25 kHz and 0.04 W/cm.sup.2) and without preservation solution: Perfusion of liver grafts (n=10) and kidney grafts (n=10) in Lactated Ringer's solution at 4° C. to wash the organs for the purpose of removing blood contained therein. After washing, the organs were stored for 8 and 24 hours for liver and kidney, respectively, and without preservation solution in the transport system with ultrasound (25 kHz and 0.04 W/cm.sup.2) between 2-6° C. [0080] GROUP 3: cold ischemia group in the transport system with ultrasound (25 kHz and 0.04 W/cm.sup.2) and with preservation solution. The group was divided into different subgroups depending on the preservation solution used. [0081] 3.1) Perfusion of liver grafts (n=10) and kidney grafts (n=10) in Lactated Ringer's solution at 4° C. The organs were then stored with Lactated Ringer's solution for 8 and 24 hours for liver and kidney, respectively, in the transport system with ultrasound at a temperature comprised between 2-6° C.; [0082] 3.2). The same as 3.1 but using Celsior solution for washing and preserving the organ; [0083] 3.3) The same as 3.1 but using UW solution for washing and preserving the organ.

[0084] Experiments were also performed to evaluate if other frequencies and/or intensities could provide more protection than what was achieved with frequencies of 25 kHz and an intensity of 0.04 W/cm.sup.2. To do this, the following experiments were performed. [0085] GROUP 4: cold ischemia group in the system with ultrasound (40, 80, 200, 580 kHz and 1 MHz) and intensity of 0.04 W/cm.sup.2 and with preservation solution: The group was divided into different subgroups depending on the frequency used. [0086] 4.1) Perfusion of liver grafts (n=10) and kidney grafts (n=10) in UW solution at 4° C. The organs were then stored in UW solution for 8 and 24 hours for liver and kidney, respectively, in the transport system with ultrasound at 40 kHz and intensity of 0.04 W/cm.sup.2 and between 2-6° C.; [0087] 4.2). The same as 4.1 but using 80 kHz and intensity of 0.04 W/cm.sup.2 and between 2-6° C.; [0088] 4.3) The same as 4.1 but using 200 kHz and intensity of 0.04 W/cm.sup.2 and between 2-6° C.; [0089] 4.4) The same as 4.1 but using 580 kHz and intensity of 0.04 W/cm.sup.2 and between 2-6° C.; [0090] 4.5) The same as 4.1 but using 1 MHz and intensity of 0.04 W/cm.sup.2 and between 2-6° C.

[0091] Experiments were also performed to verify the effect of ultrasound at an intensity greater than 0.04. The following experiments were performed for that purpose: [0092] GROUP 5: cold ischemia group in the system with ultrasound (25 kHz and intensity of 0.1 W/cm.sup.2 and with preservation solution). Perfusion of liver grafts (n=10) and kidney grafts (n=10) in UW solution at 4° C. The organs were then stored for 8 and 24 hours for liver and kidney, respectively, in the transport system with ultrasound at 25 kHz and intensity of 0.1 W/cm.sup.2 and at a temperature comprised between 2-6° C.

[0093] The following experimental groups were further added to evaluate the effect of the preservation solution and the cold conditions: [0094] GROUP 6: cold ischemia group in the conventional transport system but without preservation solution. Perfusion of liver grafts (n=10) and kidney grafts (n=10) with Lactated Ringer's solution at 4° C. to wash the organs for the purpose of removing blood contained in the organs. After washing, the organs were stored without preservation solution in the conventional cooler (without ultrasound) for 8 and 24 hours for liver and kidney, respectively, between 2-6° C. [0095] GROUP 7: non-cold condition ischemia group and combined with ultrasound (25 kHz and intensity of 0.04 W/cm.sup.2) or not. The group was divided into two subgroups. [0096] 7.1) Perfusion of liver grafts (n=10) and kidney grafts (n=10) in UW solution at a temperature between 20-25° C. The organs were then stored for 8 and 24 hours for liver and kidney, respectively, at a temperature comprised between 20-25° C.; [0097] 7.2) Perfusion of liver grafts (n=10) and kidney grafts (n=10) in UW solution at a temperature comprised between 20-25° C. The organs were then stored for 8 and 24 hours for liver and kidney, respectively, at a temperature comprised between 20-25° C. and with ultrasound at 25 kHz and intensity of 0.04 W/cm.sup.2.

[0098] Collecting and Processing Samples

[0099] At the end of ischemia, and in all the experimental groups and subgroups, liver and kidney perfusate samples were collected to assess the liver and kidney damage induced by ischemia using widely standardized techniques. Liver damage was evaluated by determining transaminase levels in the perfusate and by means of determining the caspase 3 activity in liver tissue. Kidney damage was evaluated by means of determining lactate dehydrogenase in the perfusate and caspase 3 activity in kidney tissue. MDA (malondialdehyde) levels were determined in liver and kidney tissue samples as an oxidative stress index, and ATP (adenosine triphosphate) levels were determined as an organ energy metabolism preservation index (Salahudeen A K et al., Am J Transpl 2003; 3:273-280; Omar R et al., Gut 1989; 30:510-514; Peralta et al., Am J Physiol. 2000; 279:G163-71).

[0100] The statistical study was conducted by means of an analysis of variance (ANOVA), and the level of statistical significance was then determined with a Student-Newman-Kels test.

[0101] The results obtained and shown schematically in the drawings are explained below in detail.

[0102] FIGS. 8A to 8E: show the percentage of protection, or in other words, the percentage of reduction of liver injury in biological samples consisting of liver grafts under conditions 1 to 6 described below versus injury induced by the following condition: UW solution+cold conditions (2-6° C.) when the parameters indicative of liver damage, namely, AST (aspartate aminotransferase), ALT (alanine aminotransferase), caspase 3 activity, MDA (malondialdehyde) and ATP (adenosine triphosphate), were evaluated at the end of the 8 hours of cold ischemia. Conditions (1-6): [0103] 1: UW solution, ultrasound frequency of 25 kHz and cold conditions (2-6° C.). [0104] 2: UW solution, ultrasound frequency of 40 kHz and cold conditions (2-6° C.). [0105] 3: UW solution, ultrasound frequency of 80 kHz and cold conditions (2-6° C.). [0106] 4: UW solution, ultrasound frequency of 200 kHz and cold conditions (2-6° C.). [0107] 5: UW solution, ultrasound frequency of 580 kHz and cold conditions (2-6° C.). [0108] 6: UW solution, ultrasound frequency of 1 MHz and cold conditions (2-6° C.). An ultrasound intensity of 0.04 W/cm.sup.2 was applied in all of cases 1 to 6.

[0109] FIGS. 9A to 9D: show the percentage of protection in biological samples consisting of kidney grafts in conditions 1 to 6 described below versus injury induced by the following condition: UW solution+cold conditions (2-6° C.) when the parameters indicative of kidney damage, namely, LDH (lactate dehydrogenase), caspase 3 activity, MDA and ATP, were evaluated at the end of the 24 hours of cold ischemia. [0110] 1: UW solution, ultrasound frequency of 25 kHz and cold conditions (2-6° C.). [0111] 2: UW solution, ultrasound frequency of 40 kHz and cold conditions (2-6° C.). [0112] 3: UW solution, ultrasound frequency of 80 kHz and cold conditions (2-6° C.). [0113] 4: UW solution, ultrasound frequency of 200 kHz and cold conditions (2-6° C.). [0114] 5: UW solution, ultrasound frequency of 580 kHz and cold conditions (2-6° C.). [0115] 6: UW solution, ultrasound frequency of 1 MHz and cold conditions (2-6° C.).

[0116] An ultrasound intensity of 0.04 W/cm.sup.2 was applied in all of cases 1 to 6.

[0117] FIG. 10: shows the percentage of protection in liver versus injury induced by the following condition: non use of preservation solution+cold conditions (2-6° C.)+no ultrasound when the parameter indicative of liver damage, AST, was evaluated at the end of the 8 hours of cold ischemia. [0118] 1: Lactated Ringer's solution, without ultrasound and with cold conditions (2-6° C.). [0119] 2: Celsior solution, without ultrasound and with cold conditions (2-6° C.). [0120] 3: UW solution, without ultrasound and with cold conditions (2-6° C.). [0121] 4: Lactated Ringer's solution, with ultrasound (25 KHz, 0.04 W/cm.sup.2) and with cold conditions (2-6° C.). [0122] 5: Celsior solution, with ultrasound (25 KHz, 0.04 W/cm.sup.2) and with cold conditions (2-6° C.). [0123] 6: UW solution, with ultrasound (25 KHz, 0.04 W/cm.sup.2) and with cold conditions (2-6° C.).

[0124] Note: out of the preservation solutions used, the standard solution in clinical practice is UW (University of Wisconsin) preservation solution. Lactated Ringer's solution does not contain drugs and contains only mineral salts. Celsior solution contains glutathione, and UW solution contains more drugs, such as adenosine, glutathione and allopurinol.

[0125] FIG. 11: shows the percentage of protection in kidney versus injury induced by the following condition: non use of preservation solution+cold conditions (2-6° C.)+no ultrasound when the parameter indicative of kidney damage, LDH, was evaluated at the end of the 24 hours of cold ischemia. [0126] 1: Lactated Ringer's solution, without ultrasound and with cold conditions (2-6° C.). [0127] 2: Celsior solution, without ultrasound and with cold conditions (2-6° C.). [0128] 3: UW solution, without ultrasound and with cold conditions (2-6° C.). [0129] 4: Lactated Ringer's solution, with ultrasound (25 KHz, 0.04 W/cm.sup.2) and with cold conditions (2-6° C.). [0130] 5: Celsior solution, with ultrasound (25 KHz, 0.04 W/cm.sup.2) and with cold conditions (2-6° C.). [0131] 6: UW solution, with ultrasound (25 KHz, 0.04 W/cm.sup.2) and with cold conditions (2-6° C.).

[0132] FIG. 12: shows the percentage of protection in liver versus injury induced by the following condition: UW preservation solution+without cold conditions (20-25° C.)+no ultrasound when the parameter indicative of liver damage, AST, is evaluated at the end of the 8 hours of cold ischemia. [0133] 1: UW solution without ultrasound and with cold conditions (2-6° C.). [0134] 2: UW solution with ultrasound and without cold conditions (20-25° C.). [0135] 3: Without preservation solution, with ultrasound and with cold conditions (2-6° C.). [0136] 4: UW solution, with ultrasound (25 kHz, 0.04 W/cm.sup.2) and with cold conditions (2-6° C.). Expected result: Protection 1+Protection 2=Protection (1+2) [0137] 5: UW solution, with ultrasound (25 kHz, 0.04 W/cm.sup.2) and with cold conditions (2-6° C.). Result actually observed. Protection 1+Protection 2<Protection (1+2).

[0138] FIG. 13: shows the percentage of protection in kidney versus injury induced by the following condition: UW preservation solution+without cold conditions (20-25° C.)+no ultrasound when the parameter indicative of kidney damage, LDH, is evaluated at the end of the 24 hours of cold ischemia. [0139] 1: UW solution without ultrasound and with cold conditions (2-6° C.). [0140] 2: UW solution with ultrasound and without cold conditions (20-25° C.). [0141] 3: Without preservation solution, with ultrasound and with cold conditions (2-6° C.). [0142] 4: UW solution, with ultrasound (25 kHz, 0.04 W/cm.sup.2) and with cold conditions (2-6° C.). Expected result: Protection 1+Protection 2=Protection (1+2). [0143] 5: UW solution, with ultrasound (25 kHz, 0.04 W/cm.sup.2) and with cold conditions (2-6° C.). Result actually observed. Protection 1+Protection 2<Protection (1+2).

[0144] Discussion of the Experimental Results

[0145] It is important to point out that as was expected, without applying ultrasound the temperature ranged between 2-6° C. inside the cooler, in the preservation fluid and in the organ. In addition, applying ultrasound under cold conditions (2-6° C.) did not modify the temperature inside the cooler, which was between 2-6° C. for all the experiments, but organ and preservation solution cooling was lost.

[0146] When applying ultrasound, the temperature ranged between 2-6° C. inside the cooler, the temperature of the preservation fluid ranged between 14-16° C. and the temperature of the organ between 16-18° C.

[0147] The effect of heating caused by the ultrasound is an expected result according to background documents of interest. Nevertheless, contrary to what would be expected, taking into account the vital importance of cooling the organ (4° C.) and keeping the temperature of the preservation fluid and the organ between 2-6° C. to store organs under hypothermal ischemia conditions, the results indicate that applying ultrasound does not negatively affect the effect of cooling the biological sample, and accordingly does not damage it.

[0148] Furthermore, however, it has surprisingly been verified that a very significant improvement is achieved, so much so that a synergistic effect between applying cold conditions inside the chamber of the device and applying ultrasound could be confirmed.

[0149] FIGS. 8A to 8E: as shown in these drawings, under all conditions (at different frequencies and the same intensity of 0.04 W/cm.sup.2), applying ultrasound protects the liver graft under cold ischemia conditions, said protective effects being more evident under condition 1, i.e., at a frequency of 25 kHz. Other results not shown in the drawings indicate that at higher intensities, as is the case of 0.1 W/cm.sup.2, protection of the liver graft is also obtained. For example, a percentage of protection or a reduction of injury of 55% is obtained at frequencies of 25 kHz and an intensity of 0.1 W/cm.sup.2 versus the condition: UW solution+cold conditions (2-6° C.), when the parameter for liver damage, AST, is evaluated at the end of the 8 hours of cold ischemia.

[0150] FIGS. 9A to 9D: show the same pattern of kidney protection as that shown in FIGS. 8A to 8E for the liver.

[0151] FIG. 10: as was expected, protection of the liver provided by the preservation solutions without applying ultrasound (conditions 1 to 3) is observed; protection of the liver graft is better if the UW preservation solution is used with respect to both solutions (Ringer or Celsior), and protection obtained by the Celsior solution is better than that obtained by Lactated Ringer's solution. In addition, when observing protection provided by the preservation solutions in the presence of ultrasound (conditions 4-6), unexpected results are obtained, indicating better protection of the ultrasound, but such protection is similar under all conditions (4-6). In other words, the same degree of protection is obtained regardless of the preservation solution used, whether it is Lactated Ringer's solution, Celsior solution or UW solution.

[0152] FIG. 11: shows the same pattern of kidney protection as that shown in FIG. 10 for the liver.

[0153] FIG. 12: as was expected, cold conditions without applying ultrasound (condition 1) and using the UW solution increases protection of the liver graft by about 30% when compared with preservation with UW at 4° C. and at room temperature (20-25° C.). Unexpectedly, the protection obtained by condition 2 (UW solution, in the presence of ultrasound and at room temperature) is better than that of condition 1 (UW solution and cold without ultrasound). In other words, in the presence of preservation solution at 4° C., it is better to apply ultrasound in a chamber at room temperature than under hypothermal conditions (chamber at a temperature between 2-6° C.) and without ultrasound. The results indicating that protection obtained under condition 3 (without preservation solution, under cold conditions and with ultrasound) is better than that obtained under conditions 1 and 2 are also unexpected. These results indicate that it is better to combine ultrasound and cold conditions (even without the presence of a preservation solution) than to combine the presence of a preservation solution with cold conditions (condition 1) or with ultrasound (condition 2). Conditions 4 and 5 show the expected and observed protection, respectively, when using UW solution, ultrasound and cold conditions. The expected protection would be, in the best case, the sum of protection obtained in condition 1 and of protection obtained in condition 2. In other words, due to the heat effect induced by ultrasound, protection 1+2 would not be expected to correspond to the sum of conditions 1 and 2 considered separately. However, these were not the results observed. The obtained results indicated a synergistic effect when both treatments (cold conditions and ultrasound) are combined because the protection obtained when both treatments are combined is much better than the sum of protections obtained when both treatments are applied separately. Furthermore, the protection obtained when both treatments are combined and in the presence of preservation solution is much better than that obtained when both treatments are combined without preservation solution (condition 3).

[0154] FIG. 13: shows the same pattern of kidney protection as that shown in FIG. 5 for the liver.

[0155] Accordingly, taking all the results into account, the combination of preservation solution, ultrasound and cold conditions can be an extremely efficient strategy for transport and preservation of organs during cold ischemia.

[0156] A method and equipment for transporting and storing biological samples under hypothermal conditions and without oxygen delivery under better conditions than those currently available, and with effective transmission of ultrasound to the organ or tissue inside the chamber, are provided. All this allows reducing the harmful effects of cold ischemia and increasing viability of grafts before they are implanted in the recipient, thereby preventing having to do another transplant.

[0157] The equipment and method for preservation could also be useful in secondary organs; the number of organs available for transplant could accordingly increase, thereby reducing waiting lists. Furthermore, since the injuries induced by cold ischemia during the conservation and transport of organs is reduced, the time during which organs are transported in the cooler until they are implanted in the recipient can be extended. Furthermore, the equipment is easy to transport in order to prevent, among other factors, logistic complications resulting from dynamic preservation of the organ.