Abstract:
A cell preservation system utilizes a pre-chilled coolant stored in a coolant reservoir, which is pumped at a controlled rate into a specimen vessel containing a specimen. This allows the achievement of a controlled rate of cooling the specimen and controls the formation of crystals in the specimen. The coolant reservoir is sealed, and the coolant is pumped from the specimen vessel to the coolant reservoir, whereby heat from the pump is not entered into the specimen vessel. A magnetic stirrer is selectively controlled to maintain a uniform temperature in the specimen vessel or, alternatively, is deenergized to permit temperature stratification to occur. A heater is included in the specimen vessel for precise heat control, and for raising the temperature of the coolant in the specimen vessel to a value suitable for beginning a cooling cycle.

Description:
BACKGROUND OF THE INVENTION 
     The present invention relates to techniques for preserving a biological cell specimen. 
     The preservation of viable cell specimens is a very important field of science. It is essential to long-term preservation of tissue specimens, sperm, and fertilized ovum (zygotes), among others. Cooling of cell specimens to cryogenic temperatures, and then holding the specimens at cryogenic temperatures is typically used for preservation. The cooling must be carefully controlled to minimize crystal size so that crystals are prevented from attaining sizes which are capable of damaging cells. During cooling, the temperature range of from room temperature to about -40 to -50 degrees Celsius is critical for crystal formation. Precise control of cooling in this range is essential to the viability of the specimen. The most critical temperature range is from about zero degrees C to a few degrees below -8° C. In this range, a specimen releases heat of fusion during cooling. Thus, in this range, the amount of heat which must be removed to maintain a programmed cooling gradient increases markedly. As a consequence, the rate of heat removal requires precise control to maintain a desired cooling rate through this range. Once the speciment is cooled below about -40°  or -50° C., crystal formation is no longer a problem, and thus less precise control of the cooling rate is permitted. 
     The cooling parameters for different lines of cells may differ. Thus, the cooling programs (temperatures, rates and times) for different cell lines may also differ. 
     At the present, two ways to achieve the cooling of the cells are employed: cooling with a liquified gas (typically liquid nitrogen) and cooling by mechanical refrigeration. 
     When liquid nitrogen is employed, it is possible to achieve a very high temperature gradient. For example, an initial cooling rate of as much as 80° C. per minute is possible. The specimen is usually dipped in a container with liquid nitrogen. Although this gives a large cooling rate, the cooling rate cannot be controlled. Also, liquid nitrogen presents a problem in handling. 
     Better control of cooling is provided using vapor from a liquified gas (liquid nitrogen). The specimen to be cooled is placed in a vessel with the liquid nitrogen, but out of physical contact with the liquid. The cold vapor coming from the liquid nitrogen is relied on for cooling. This method suffers from the low thermal capacity of a gas. As a consequence, it is difficult to maintain uniform cooling rates and temperatures across a specimen. 
     Mechanical cooling employs a refrigeration unit that uses coolant coils about the walls of a specimen container. A heat transfer medium, such as, for example, ethanol, in the specimen container aids in the transfer of heat from the specimen to the coolant coils. This technique does not need liquid nitrogen, and can provide cooling down to at least -80° C. However, the temperature gradient attainable by mechanical refrigeration is limited to about 2-3 degrees Celsius per minute. For many biological specimens, this rate is not great enough to prevent the formation of crystals of damaging size through the critical temperature range above -40° C. 
     OBJECTS AND SUMMARY OF THE INVENTION 
     It is an object of the invention to provide controlled cooling of a specimen and the ability to achieve high temperature gradients. 
     It is a further object of the invention to provide a controlled cooling system which does not require the handling of liquified gas. 
     Briefly stated, a cell preservation system utilizes a pre-chilled coolant, stored in a coolant reservoir, which is pumped at a controlled rate into a vessel containing a specimen. This allows the achievement of a controlled rate of cooling the specimen and controls the formation of crystals in the specimen. 
     According to an embodiment of the invention, there is provided a system for cooling a specimen comprising: a coolant, a specimen vessel, a coolant reservoir, a coolant in the specimen vessel and the coolant reservoir, means for circulating the coolant between the specimen vessel and the coolant reservoir, means for cooling the coolant in the coolant reservoir, and means for controlling the means for circulating to attain a predetermined rate of cooling of the specimen in the specimen vessel. 
     According to a feature of the invention, there is provided a process for achieving a controlled cooling of a specimen, comprising: placing a specimen to be cooled in a specimen vessel, prechilling a coolant in a coolant reservoir, circulating the coolant through the coolant reservoir and the specimen vessel at a pumping rate effective for cooling the specimen at a predetermined cooling rate, sizing the coolant reservoir to provide sufficient heat capacity to attain cooling at the predetermined rate. 
     The above, and other objects, features and advantages of the present invention will become apparent from the following description, read in conjunction with the accompanying drawings, in which like reference numerals designate the same elements. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a block diagram of the system of the present invention. 
     FIG. 2 is a schematic drawing of an embodiment of the system of the present invention. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Referring to FIG. 1, there is shown, generally at 10, a block diagram of a cell preservation system 10 according to an embodiment of the invention. A specimen vessel 12, for containing a specimen to be cooled, is placed in fluid communication with a coolant reservoir 14 which contains a pre-chilled coolant. The specimen vessel is as seen particularly in FIG. 2, an open top component so coolant present in the specimen vessel is exposed to the ambient environment and hence, coolant present in the specimen vessel is at the same pressure as exists in the ambient environment. The coolant is pre-chilled by refrigeration system 16 communicating with coolant reservoir 14. Coolant reservoir 14 is a sealed reservoir filled with a coolant. The coolant is pumped by pump 18 from a location near the top of specimen vessel 12 into coolant reservoir 14. Since coolant reservoir 14 is sealed, the entry of coolant into the top thereof forces prechilled coolant from the bottom of coolant reservoir 14 to specimen vessel 12. The coolant then exchanges heat with the contents of specimen vessel 12. The initial temperature and heat capacity of the coolant in coolant reservoir 14, and the rate at which pump 18 circulates the coolant, determines the cooling rate of a specimen in specimen vessel 12. That is, if pump 18 is stopped, cooling of a specimen in specimen vessel 12 is limited by the temperature and heat capacity of a heat transfer medium existing in specimen vessel 12. If pump 18 is operated at high speed, substantially the full heat capacity of coolant reservoir 14 is available to cool the specimen. At intermediate speeds of pump 18, intermediate rates of cooling are attained. The pumping speed of pump 18 is controlled by a controller 20. 
     It is to be noted that pump 18 pumps coolant from a location near the top of specimen vessel 12. Coolant is forced into specimen vessel 12 near its bottom. This has a number of desirable effects. First, any friction heat generated by pump 18 is carried to coolant reservoir 14, rather than to specimen vessel 12. Also, removing coolant from the top of specimen vessel 12, and entering fresh precooled coolant in the bottom of specimen vessel 12, permits stratification of the temperature of the coolant in specimen vessel 12, as will be described hereinafter. 
     Referring to FIG. 2, cell preservation system 10 includes a coolant 24 in specimen vessel 12. Coolant reservoir 14 is filled with coolant 24 which is pre-cooled by refrigeration system 16. Any suitable coolant can be used in the system of the present invention. The coolant must be of a type that remains fluid at the lowest temperature experienced in the apparatus and must be benign to a specimen placed therein. Examples of suitable coolants include ethanol and silicone oil. A specimen container 26, containing a specimen to be cooled, is placed in specimen vessel 12. Specimen container 26 is surrounded by coolant 24 which provides the cooling. The cooling rate is controlled by changing the speed of pump 18 which, in turn, is controlled by controller 20. 
     As is conventional, besides a specimen, specimen container 26 may also contain a cryo-protectant fluid. Since the use of a cryo-protectant fluid is conventional, further discussion thereof is unnecessary. 
     To insure efficient heat exchange between a specimen in specimen container 26 and coolant 24, pre-chilled coolant 24 is delivered to specimen vessel 12 through an inlet pipe 34 located at the bottom of specimen vessel 12. The coolant, heated by contact with specimen container 26, exits specimen vessel 12 through outlet pipe 36 located at the top of specimen vessel 12. To provide a uniform temperature inside the volume of coolant 24, a magnetic stirrer 30 is placed inside specimen vessel 12. Magnetic stirrer 30 is a ferromagnetic mass which may be free inside specimen vessel 12, or may be retained in a bearing for guiding rotation thereof. A permanent magnet 31, outside vessel 12, is rotated by a stirrer motor 32. Magnetic coupling between permanent magnet 31 outside specimen vessel 12, and the ferromagnetic material making up magnetic stirrer 30 inside specimen vessel 12, rotates magnetic stirrer 30 to attain a substantially uniform temperature of coolant 24 therein. 
     Controller 20 controls the rate at which pre-chilled coolant 24 is delivered into specimen vessel 12. Therefore, if it is desired to increase the rate of cooling in specimen vessel 12, the pumping speed of pump 18 is increased. This allows the achievement of any desired rate of cooling. In addition, the cooling rate can be tailored for different specimens, or can be tailored for different rates at different stages of cooling a particular specimen. 
     It will be noted that magnetic stirrer 30 maintains a substantially uniform temperature in coolant 24 throughout the interior of specimen vessel 12. At some point in a cooling program, it may be desirable to stop magnetic stirrer 30 to attain maximum cooling of a specimen. This permits temperature stratification to take place in specimen vessel 12. That is, the colder coolant 24, entering near the bottom of specimen vessel 12, is permitted to displace warmer coolant above it so that specimen container 26 is immersed in coolant 24 at substantially the temperature at which it leaves coolant reservoir 14. 
     Controller 20 may operate open loop. That is, it may operate without feedback informing it of the temperature actually attained in the vicinity of specimen container 26. In one open-loop embodiment, controller 20 contains a microprocessor which controls the pumping rate of pump 18 according to a preset time schedule. The time schedule may be selectable by operator controls according to the type or size of the specimen in specimen container 26. Alternatively, closed-loop control may be achieved using, for example, a conventional temperature probe 38. 
     Another feedback technique which may be used instead of, or in addition, to temperature probe 38, includes the provision of a dummy specimen container 46 having a temperature probe (not shown) therein. Signals from the temperature probe in dummy specimen container 46 are connected to controller 20. Dummy specimen container 46 preferably contains materials having a temperature and heat capacity closely resembling those of specimen container 26. Thus, the temperature inside dummy specimen container 46 can be expected to follow closely the temperature in specimen container 26. 
     It is also within the contemplation of the inventor that a temperature probe (not shown) may be inserted into specimen container 26 to measure the temperature therein directly. 
     Specimen vessel 12 preferably includes a layer of insulation 40 about its exterior to reduce heat loss. Similarly, coolant reservoir 14 includes a layer of insulation 42 about its exterior. Cooling coils 44 are disposed about the exterior of coolant reservoir 14 beneath insulation 42. Alternatively, cooling coils 44 may be disposed inside coolant reservoir 14, either affixed to an inside surface, or suspended within coolant 24. 
     The size of coolant reservoir 14, the size of specimen vessel 12, the cooling capacity of refrigeration system 16, the amount and rate of heat removal from specimen container 26, and the desired throughput of cell preservation system 10 are interactive parameters. The temperature of coolant 24 in coolant reservoir 14 must remain low enough through the end of a cooling cycle to attain the desired cooling rate. Thus, the initial quantity of coolant 24 in coolant reservoir 14, and its initial temperature must be such that coolant 24 in coolant reservoir 14 has sufficient heat capacity to absorb all of the heat required from a specimen in specimen container 26 to reduce the temperature of the specimen to the desired value, and to do so at the desired cooling rate. 
     The throughput (number of specimens cooled per hour) is limited by the cooling capacity of refrigeration system 16. In normal operation, refrigeration system 16 operates continuously. During the cooling of a specimen, the temperature of coolant 24 in coolant reservoir 14 rises since refrigeration system 16 is incapable of removing heat as fast as required to attain the desired cooling rate of the specimen. After cooling one or more specimens, the temperature of coolant 24 in coolant reservoir 14 may rise to a value at which effective cooling of a further specimen is not possible. A chilling cycle is then required to decrease the temperature of coolant in coolant reservoir 14 to a value permitting the cooling of further specimens. 
     In combination, the size of coolant reservoir 14, the size of specimen vessel 12, the control of the pumping of pump 18, the cooling capacity of refrigeration system 16, and the throughput of specimens are interactive parameters. Once these interactive parameters are understood, and cell preservation system 10 is created in accord therewith, controlled cooling of a specimen at any desired rate is possible without incurring the drawbacks of liquid nitrogen coolant. 
     It will be understood by one skilled in the art that permanent magnet 31 is merely symbolic of a device for producing a rotating magnetic field effective for moving magnetic stirrer 30 inside specimen vessel 12. Other techniques for magnetically driving magnetic stirrer 30 should be considered to fall within the scope of the invention. For example, a plurality of magnetic coils (not shown) may be substituted for permanent magnet 31 and stirrer motor 32. Controller 20 may provide signals for energization of the magnetic coils to provide a moving magnetic field in whose influence magnetic stirrer is forced to move. Preferably, a rotating magnetic field is provided. This should not be taken to exclude a reciprocating magnetic field for some purposes. 
     Precision of control during cooling may be enhanced by the availability of controlled addition of heat during certain stages. In addition, it may be desirable to raise the temperature of coolant 24 in specimen vessel 12 at the completion of the cooling of one specimen in preparation for the installation of the next specimen. An electric heater 48 is provided optionally on the interior of specimen vessel 12 for these and other purposes. Electric heater 48 may be manually controlled, but is preferably controlled by controller 20. 
     The presence of electric heater 48 also permits the apparatus to be used for controlled heating of specimens. 
     Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.