The present invention concerns a thermoregulated refrigerating container, more specifically a refrigerating container intended for the transportation of organs for transplantation.

The treatment of an organ after harvesting during its transportation and during its storage in conditions which will optimise its reimplantation into the recipient are important practical problems for surgical teams involved in organ transplantation. A longer preservation time would allow a better distribution of organs between transplantation centres and consequently a more appropriate selection of recipients. Furthermore it would allow an increase in the preparation time of a given recipient. In liver transplantation particularly, the possibility of preserving an organ for several hours of cold ischemia has allowed the development of the techniques of "reduced liver" for children, and of "split livers" in which one organ is divided and used for two recipients.

Over the last few years signif cant advances in the field of organ preservation have concerned mainly the improvement of preservation solutions. For abdominal organs the Belzer solution has replaced the Eurocolllns preservation solution. The Belzer solution allows an increase in the preservation time, even if it does not appear to be significantly better than the Eurocollins

for short periods of cold Ischemia. With the Belzer solution a kidney can be preserved for about 40 hours, a liver for about 18 hours and a pancreas for about 34 hours, without significantly Increasing the incidence of primary non-function of the graft or of vascular thrombosis.

Recent efforts in improvement of preservation solutions has not been matched by equal attention to the thermal conditions during the period after harvesting and during the transportation of organs in the ice box, in spite of the fact it has been shown in the laboratory that organs transported in boxes filled with preservation solutions and resting on crushed ice, inside an ice box, showed core temperatures varying between 0.3°C and 7.8 ^β C according to the kind of packaging and to the extent to which the organ was covered with ice.

It has also been observed that the physiological conditions of the organ to be transported are modi ied by the preservation temperature, the optimal temperature varying to some extent according to the kind of organ but generally being between +2 ^β C and +7°C. Furthermore it is Important that the preservation temperature is kept as steady as possible. There is experimental evidence that the physiological conditions of the organ are influenced by the speed of cooling after harvesting, which should neither be too fast nor too slow. It is therefore necessary to standardise the process of cooling of the organ, and to store it in a refrigerating box where the

temperature is constant, and can be monitored and adjusted.

Several models of thermoregulated transportable cooling boxes are presently used by transplantation teams or have been described in the prior art. Some boxes, described for instance in DE-A-2 732 321 or in DE-A-3 639 089, make use of ther o-electrical elements such as Peltier blocks to cool the inside of the thermo-regulated enclosure. These devices need however an outside electrical source, and cannot be independent of it. A cylindrical box for organ transplantation (diameter 480 mm, height 570 mm) sold under the name of TRANSPLANTHERM (Registered Trade Mark) uses a thermostat jacket made of two communicating parts: one part, in the cover, contains ice and a second part, inside the lateral walls, contains water. A circulation can be established between the two parts to obtain the desired temperature. The device can function for 20 hours without an external energy source. The working temperature has to be determined beforehand and the respective amounts of water and ice need to be calculated by weighing them according to tables that take into account the temperature of the water used. The preparatory manipulation of the water jacket, and of the amounts of the crushed ice and the water, which have to be exactly predetermined according to the desired temperature and of the temperature of the water, are very demanding. Furthermore it is not possible to change the temperature inside the box, and no

modification of the speed of cooling of the organ can be made once the device has been prepared.

A first aim of the invention is to lower as rapidly as desired the temperature of the harvested organ, which is initially at a temperature of 15 ^β C to 10"C, to the desired temperature, between 2 ^β C and 7 ^β C, usually 4°C.

A second aim of the invention is to keep the desired temperature of the enclosure, and therefore of the organ, constant with high accuracy, e.g. of ±0.1 ^β C, while having the possibility of modifying the temperature during the period of preservation.

A third aim of the invention is to have independence as long as possible, preferably of the order of 30 hours at 4 ^β C, without the need of an external power source, or of a renewed supply of cooling material such as crushed ice or similar, which may not be readily availabl .

A further aim of this invention is to avoid the loss of the organ in the event of a ault in the functioning of the thermo-regulated refrigerated container, and to enable emergency measures to be taken even in the absence of an external source of electrical power or of cooling material. These aims are reached in preferred embodiments of the invention. It has been observed, quite surprisingly, that if a layer of crushed ice is placed at the bottom of a refrigerated container, and this container is left undisturbed and without any system of regulation or

ventilation, in an ambient atmosphere with a room temperature in the region of 20 ^β C to 25 ^β C, an atmosphere inside the container is established in which the temperature varies between a narrow cold zone near the ice and a large warmer region above. The temperature variation between the ice and the cover at the top does not change linearly but is very rapid in the proximity of the ice, and very slow above a level of about 40 mm from the ice. We have measured for instance:

Another example of the measurement of the internal temperature in such a box is given in ^" Figure 1.

In the invention, however, the container is equipped with one or more internal mixing devices, especially fans, to distribute and mix the atmosphere within the container.

In use, the organ will usually be placed in an Intestinal bag or a protecting box and be bathed in preservation fluid. To keep the organ at a steady temperature it would be better to place it in the upper region inside the container at a distance of at least 30 mm to 40 mm above the surface of the crushed ice.

Thanks to a fan or other mixing device the air inside the container can be mixed so that it passes over the ice and its temperature be decreased to the desired value. In a preferred embodiment two fans are preferably fitted on the interior surface of a cover of the container, one blowing the air flow towards the crushed ice, the other aspirating it, so that their contained efficiency is increased. Then, the desired temperature, 4 degrees for instance, can be reached in about 15 minutes. This temperature remains steady after the fans are stopped for a while, before increasing slowly. When the temperature increases above a predetermined value, the fan(ε) come into function again. One or more temperature probes, coupled to an electronic system of detection and regulation, turn the fan(s) on and off, according to whether the desired temperature has been reached, or has returned to a slightly higher value, the differential being, for instance 0.1°C.

The desired values, the physical parameters of the atmosphere within the container, as well as information concerning the functional state of the container, can appear on an external display.

One embodiment of the invention will now be described with reference to the accompanying drawings in which:

Figure 1 shows a temperature curve, in a steady state, without regulating mechanism, as measured at different heights between the surface of crushed ice and

the top of a container;

Figure 2 shows the temperature changes during cooling, of the atmosphere of the enclosure and of the liquid in which the organ is bathed respectively, according to whether fans are driven by a probe sited in the atmosphere within the container (interrupted line) or by a probe sited in the preservation solution (continuous line);

Figure 3 shows a vertical cross section of a preferred embodiment of container; and

Figure 4 is a circuit diagram for electronic control means.

The refrigerated container is made of an insulating box 1 in the shape of a rectangular pa allelepiped. This box has a double plastic wall 2, this structure and its fabrication being known per se. The capacity of the container is approximately 48 litres. In the lower third of the container, two inner facing walls are constructed with notches 3 and/or shelves 4 so that they can hold four removable bars 5 which fit into the notches, or a grid resting on the shelves. A holding system with six eyelets 6 is welded to the bottom of the box which allows belts 7 to steady a box 8 containing the organ on the removable bars 5 or the grid. The base of the box holds a reservoir 9 for the water from melted ice, the capacity of which is about 7 litres, equipped with an outflow tap 10 and a screw cap, which permits the washing of the reservoir. The

separation of the ice 11 from the water from melting increases the self-contained nature of the container.

On the lower, inner, side of a cover 12 are fixed two fans 13, 14, for which power is provided by two batteries 15, 16 incorporated into the cover. The blades of one of the fans are oriented in such a way as to propel the air downwards towards the surface of the ice 11 at the bottom of the container, the blades of the other are oriented in the opposite way, so as to aspirate the air towards the top. The two fans working together mix the air in a homogeneous fashion, so that the desired temperature can be rapidly obtained. Two temperature probes 17, 18 are removably held in a stand also fixed to the interior of an inner surface of the cover 12, to allow the measurement of the inside temperature at to ±O.l'C. Each probe contains a negative temperature coefficient (NTC) resistance to measure the temperature. One, 17, is to allow display of the temperature, the other, 18, is to regulate it. Furthermore, a magnetic device (not shown), sited inside the cover, detects opening of the cover, in order to Interrupt the measurement and detection circuitry as well as the power supply to the fans. On the top of the cover is an electronic control device 19, connected to the fans and the probes. It allows a continuous reading of the inside temperature, setting of the desired temperature, and giving warning alarms for high and low temperatures (for instance, desired temperature 4 ^β C, high

temperature warning 5 ^β C, low temperature warning 3 ^β C). The appliance includes- a timer to count the time from for instance the time at which the apparatus has begun to operate, or the time at which the organ has been introduced into the container. The timer is preferably supplied by a different battery than the one supplying the rest of the apparatus. The electronic appliance also displays the remaining life of the battery supplying the fans. By means of an appropriate connection, known per se, it is possible to recharge the batteries and to supply the electronic appliance through the cigarette lighter of an ambulance or helicopter or by a battery charger connected to the mains.

Figure 4 shows a schematic diagram of the electronic detection circuit. Control is given by an integrated circuit LM 3914N (NATIONAL SEMICONDUCTOR). The assembly includes an adjustable bridge to determine the scale of measurement, and a sensor bridge containing a first NTC resistance (e.g. of 40,4 kΛ at 25 ^β C); it is the value of the measured voltage (pin 5) as compared to the two reference voltages (pin 3, 4 and 6) that determines the active point of the scale, when this point is passed, the result is the turning on of a relay that sets the two fans into motion (pin 5 and 3, connected). A polarised capacitor of 470 μf is connected in parallel to the pins supplying the relay so as to avoid instability of the system when the temperature value is close to the desired temperature. This permits

the lowest possible consumption of electrical power. Satisfactory functioning of the device depends on the choice of the two resistance values of the two potentiometers. The resistance R will be chosen as close as possible to the NTC resistance at the middle of the temperature range that will have to be measured (R is adjustable; the current applied to the relay is given by the formula 1=12.5/R and a value of about 30 mA will be chose ) . The choice of the desired temperature can be made between 2 ^β C and 8°C by steps of 1", by changing the resistance values (at 2°C, R=115,8 kΩ; at 3°C, R=lll,3 kΩ; at 4°C, R=107,7 kΩ; at 5 ^β C, R=103,l kΩ; at 6 ^β C, R=97,7 kΩ; at 7°C, R=94,7 kΩ; at 8°C, R=90.5 kΩ; at 9°C, R=88,7 kΩ). A rotatable control showing the desired temperature will be used.

The consumption of the integrated circuit itself is 2.0 mA. The recommended supply voltage between 6 V - 15 V does not entail a detection error greater than ±0.1°. The 22 nf decoupling capacitor means that shielded wires need not be used. Power is supplied by two lead batteries connected exchangeably in parallel. They are rechargeable up to 1200 cycles, allowing a useful life of 5 years at a working temperature between -2"C and _+50*C. It will be noticed that the stability of the supply voltage is not critical, as a very effective regulator is already provided in the LM3914N. It will also be noticed that the circuit can either be permanently left switched on or be started at the time of measuring, as no

stabilisation period is needed, even if the NTC may require a few seconds to equilibrate its temperature to its surroundings.

A battery charge display is included in the electronic appliance 19 located on the cover and shows three LED fields respectively green, yellow and red, showing the state of charge of the battery being used. The battery charge detector can be calibrated in such a way as to show the remaining life, for instance the green field meaning 12 to 17 hours of remaining functioning time, the yellow one from 9 to 1, and the red the need to exchange immediately onto the second battery.

The time and/or temperature display can be coupled to a warning alarm. The functioning of the apparatus will now be described, starting from a warning that an organ harvesting operation could possible take place:

The charge of the two batteries is first checked. If one is on the yellow indicator, it should be charged. The full charge takes approximately 5 hours, and a half- charge approximately 1 hour. The maximum working time for each battery is 20 hours for a full charge and 10 hours for a half charge.

The venting tap 10 should be closed. Before departure for the harvesting, the container is loaded with approximately 15 litres of crushed ice 11. It is not necessary to start the temperature regulation system at this time. About 1 hour before the organ is placed

into the container, the temperature regulation system is put into action by setting the desired temperature between 2*C and 7 ^β C by means of a temperature dial. It is checked at this moment that the level of the crushed ice is just below the support bars 5 or grid. When the organ is deposited into the container, it should be checked that its box 8 is well centred, so that air can circulate freely around it. The box 8 is anchored on the supporting bars 5 by means of three elastic belts held by the eyelets 6- Once the cover 12 is closed, the time counter is started.

Figure 2 shows the cooling curves of the enclosure (a) and of the organ (b), when the cooling phase is driven in accordance with the temperature of the enclosur .

To increase the speed of cooling, the regulating probe can be placed in the preservation luid in the box containing the organ, until the desired temperature has been reached. Cooling of the organ will occur according to the curve (d), while the temperature of the enclosure will decrease to about 1.5"C (curve σ). It is however preferable, if this method is chosen, not to dissociate the two probes, and to place the measurement and display probe 17 in the preservation fluid too, so to avoid a faulty interpretation of the values on the display. In this case a box equipped with two openings specifically designed to accept the probes will preferably be used. When the desired temperature has been reached, the

temperature in the box can be regulated either from the temperature of the preservation fluid, or from the temperature of the enclosure, replacing the probes in their stands. Regulation at ±0.1" from the desired value is achieved by mixing the air under the in luence of the two fans 13, 14 fixed on the inside of the cover. The f ns come into action as soon as the temperature is higher than the desired value, and stop as soon as this is reached. So as to obtain the longest possible working time, the contact between the crushed ice and the water from melting is avoided by means of the reservoir 9 which is separated from the inside of the container by a double floor, which can be constructed as a perforated grid. In the event of a break-down in the regulation system it will be sufficient in order to keep the organ at a low temperature to take out the removable bars 5 from the notches and lay them on the ice 11, and to put the box containing the organ on top of them at about 10 mm from the sur ace of the crushed ice or even directly on the ice, so as to minimise the damage suffered by the organ. It: will be noticed that the thermal exchange between the box and the ice is less efficient in the absence of water.

It will also be noticed that all thermal exchanges within the box involve only small amounts of energy; exchange between warm air and cold air and exchange between the air and the box containing the organ. If the features of the container are to be best exploited, in

particular during the initial phase of cooling of the organ, the box containing the graft will preferably be made of stainless steel, rather than of polypropylene as is usually the case. Furthermore, to avoid the risk of an excessive cooling of the organ, i.e. below -l ^β C, only ice (temperature approximately 0 ^β C) will preferably be used, and not cooling elements or the like, as used in freezers.

It will also be noticed that in the embodiment described above, all of the electronic and electromechanical devices are fixed inside, within or above the cover, so as to avoid the passing of wires between the different parts of the container, which minimises wear. It is however self evident that other embodiments in which the fans, the batteries, and the electronic elements are placed differently, on the lateral wall for instance, are possible. It is equally self evident that variants in the construction of the container, whether concerning its shape (cylindrical, for instance), of the materials used for insulation, of the external protective elements, whether made of metal or of other substances, and also a construction in two separate units to allow the separation of the water reservoir from the body of the container are possible. The above described electronic circuitry is given as an example of a simple and reliable appliance, but could be replaced by any other equivalent circuitry.

The two probes each containing a NTC resistor which

are easily available on the market could be replaced by a single probe containing two NTC resistors. Alternatively there may be two probes containing two NTC resistors each. Each probe could then provide at the same time for measurement, display and regulation of the temperature, in this variant one of the probes can be permanently placed into the enclosure, for instance fixed inside the cover ( "enclosure probe" ) . The other probe with two NTC resistors can be moved inside the enclosure, and in particular so that it can be fixed onto or inside the box containing the preservation liquid and the organ ("organ probe"). By means of a switch, the regulation of the container can be driven either from the organ probe or from the enclosure probe. In fact it is possible to drive the organ cooling phase from the organ probe, and the following time during the journey from the enclosure probe. One probe can replace the other in the event of a fault.

The refrigerating thermo-regulated container embodying the invention has been developed mainly for the transportation of organs. The man skilled in the art will observe that it is equally suitable for the storage and transportation of other biological products the stability and integrity of which are affected by temperature, such as blood, protein solutions, vaccines and biological reagents in general.