US20060260328A1

US20060260328A1 - Cryogenic biological preservation unit with active cooling and positive atmospheric seal lid

Info

Publication number: US20060260328A1
Application number: US11/130,132
Authority: US
Inventors: Bryce Rampersad
Original assignee: Praxair Technology Inc
Current assignee: Praxair Technology Inc
Priority date: 2005-05-17
Filing date: 2005-05-17
Publication date: 2006-11-23
Also published as: WO2006125060A3; WO2006125060A2; TW200718356A

Abstract

A cryogenic biological preservation unit and method of operation wherein the atmosphere within the unit is sealed from the outside atmosphere by pressure maintenance provided by varying amounts of refrigeration from a cryocooler and by a sealed lid employing pliable polymeric material maintained in compression.

Description

TECHNICAL FIELD

This invention relates generally to preservation of biological samples and, more particularly, to preservation of biological samples at cryogenic temperatures.

BACKGROUND ART

There is a growing trend toward cryogenic storage of biological samples at temperatures below 140 K. This trend is driven by the fact that little to no sample degradation occurs below the sample glass transition temperature which is about 140 K. Conventional cryogenic biological sample preservation units that store biological samples at temperatures below 140 K use liquid nitrogen to keep the biological samples cold. These units typically store the samples within a vacuum insulated space above a pool of liquid nitrogen or immersed within the pool of liquid nitrogen. The liquid nitrogen needs to be periodically replenished due to loss of nitrogen vapor from the unit. This is costly, not only because of the cost of the nitrogen, but also because of the complicated procedures required to handle the liquid nitrogen. Moreover, there is another problem caused by contaminant gas migration into the unit.

SUMMARY OF THE INVENTION

One aspect of the invention is:
A cryogenic biological preservation unit comprising:
(A) an insulated vessel having a vessel interior, a lid positioned within an opening allowing access to the vessel interior, and a seal comprising pliable polymeric material in contact with the lid;
(B) a cryocooler positioned to provide refrigeration to the insulated vessel; and
(C) a pressure relief conduit having a pressure relief valve and being in communication with the vessel interior.
Another aspect of the invention is:
A method for operating a cryogenic biological preservation unit comprising:
(A) providing refrigeration from a cryocooler to the interior of an insulated vessel, said insulated vessel having a lid and a seal comprising pliable polymeric material in contact with the lid;
(B) maintaining the pliable polymeric material in compression; and
(C) maintaining the pressure within the interior of the insulated vessel within 2 psi of the pressure outside of the insulated vessel.
As used herein the term “cryocooler” means a refrigerator which can produce refrigeration below 193 K for the purpose of cooling biological samples.
As used herein the term “cold head” means the portion of the cryocooler containing the cold heat exchanger, aftercooler and regenerator.
As used herein the term “cold finger” means a portion of a cold head that is configured such that the cold heat exchanger is located at one end of the cold head. The cold finger refers to the portion of the cold head with this configuration that, in operation, is at a temperature below that of the aftercooler.
As used herein the term “biological sample” means an organic material. Some examples of biological samples are proteins, blood platelets, cartilage and heart valves.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross sectional representation of one preferred embodiment of the cryogenic biological preservation unit of this invention.
FIG. 2 is a representation of one preferred sealing system which may be used in the practice of the cryogenic biological preservation unit of this invention.

DETAILED DESCRIPTION

The invention will be described in detail with reference to the Drawings. Referring now to FIG. 1, there is shown a cryogenic biological preservation unit comprising an insulated vessel having a vessel wall 1 and having insulation, typically vacuum insulation, 3 adjacent the inside of vessel wall 1. Vessel wall 1 and insulation 3 define the vessel interior or storage space 2. In the lower portion of vessel interior 2 is a pool of liquid nitrogen 4.
Within vessel interior 2 and preferably above liquid nitrogen pool 4 there is stored at least one biological sample. In FIG. 1 there is illustrated in representational form a plurality of biological samples 5 on a rack system. In general the cryogenic biological preservation unit of this invention will have a diameter within the range of from 30 to 60 inches and a height within the range of from 45 to 75 inches. Depending upon the size of the biological samples and upon the type of rack system used, the cryogenic biological preservation unit of this invention can accommodate or store up to 15,000 to 80,000 biological samples in 1-2 ml plastic vials. Large items such as blood bags and organs can also be stored.
The cryogenic biological preservation unit of this invention has an opening 25 which allows access to the vessel interior 2 from outside the vessel and through which biological samples are put into and removed from the vessel interior. Within opening 25 there is positioned sealed lid 7 which is typically insulated using a closed cell foam such as expanded polystyrene, and which is positioned in opening 25 when access to vessel interior 2 is not desired. Sealed lid 7 will be described in greater detail below.
Any suitable cryocooler may be used in the practice of this invention. Among such cryocoolers one can name Stirling cryocoolers, Gifford-McMahon cryocoolers and pulse tube refrigerators. A pulse tube refrigerator is a closed refrigeration system that oscillates a working gas in a closed cycle and in so doing transfers a heat load from a cold section to a hot section. The frequency and phasing of the oscillations is determined by the configuration of the system. The driver or pressure wave generator may be a piston or some other mechanical compression device, or an acoustic or thermoacoustic wave generation device, or any other suitable device for providing a pulse or compression wave to a working gas. That is, the pressure wave generator delivers energy to the working gas within the pulse tube causing pressure and velocity oscillations. Helium is the preferred working gas; however any effective working gas may be used in the pulse tube refrigerator and among such one can name nitrogen, oxygen, argon and neon or mixtures containing one or more thereof such as air.
The oscillating working gas is preferably cooled in an aftercooler and then in a regenerator as it moves toward the cold end. The geometry and pulsing configuration of the pulse tube refrigeration system is such that the oscillating working gas in the cold head expands for some fraction of the pulsing cycle and heat is absorbed by the working gas by indirect heat exchange which provides refrigeration to the vessel interior. Preferably the pulse tube refrigeration system employs an inertance tube and reservoir to maintain the gas displacement and pressure pulses in appropriate phases. The size of the reservoir is sufficiently large so that essentially very little pressure oscillation occurs in it during the oscillating flow.
The cryocooler components 10 include the mechanical compression equipment (pressure wave generator), the inertance tube and reservoir, the final heat rejection system and the electrical components required to drive and control the cryocooler. Electrical energy is primarily converted into acoustic energy in the pressure wave generator. This acoustic energy is transferred by the oscillating working gas to the cold head 8 via the transfer tube 9. The transfer tube 9 connects the pressure wave generator to the aftercooler located at the warm end of the cold head 8, where heat is removed as previously described. The cryocooler can be controlled to provide varying amounts of refrigeration to the cold end of the cold finger 6 depending on the conditions in the cryogenic biological preservation unit vessel interior 2. This is accomplished by modulating the acoustic power output from the pressure wave generator by varying the voltage and thus the electrical power supplied. The cryocooler would preferably be controlled based on the temperature of the vessel interior 2 of the cryogenic biological preservation unit.
In the embodiment of the invention illustrated in FIG. 1, cold finger 6 penetrates into vessel interior 2 and provides refrigeration directly to the vessel interior. The refrigeration cools and condenses nitrogen vapor within the upper portion of the vessel interior 2 as will be more fully described below, thus eliminating the need to replenish the liquid nitrogen from outside the unit and thereby minimizing costly and complicated liquid nitrogen handling procedures and systems. The condensed nitrogen falls by gravity to the liquid nitrogen pool 4 in the lower portion of the vessel interior.
The temperature at the lowest level of the sample storage within the vessel interior may be as low as 77 K and is generally within the range of from 80 to 95 K. However, the normal temperature at the upper levels of the sample storage may be within the range of from 95 to 140 K without the use of the integrated cryocooler of this invention. Samples in the top racks of conventional cryogenic biological preservation units could exceed the glass transition temperature of the biological samples when the lid is removed for access to the interior. For this reason, storage of biological samples in the upper portion of conventional cryogenic biological preservation units is often avoided. However, with the cryogenic biological preservation unit of this invention which provides cryocooler refrigeration to the upper portion of the vessel interior, biological samples may be stored in the upper portion of the vessel interior without fear of degradation due to elevated temperature. This increases the effective capacity of the unit which is another advantage of the cryogenic biological preservation unit of this invention over conventional systems. In the practice of this invention, the cryocooler will continuously recondense all or most of the nitrogen vaporized due to heat leak caused by opening and closing the vessel and by the integration of the cryocooler with the vessel itself.
In the practice of this invention the pressure within the interior of the insulated vessel is maintained within 2 pounds per square inch (psi) of the pressure outside of the insulated vessel. In this way the sealed lid will prevent nitrogen vapor from flowing out from the vessel interior past the lid, and also will prevent ambient gas from flowing into the vessel interior past the lid. This serves to reduce or even eliminate nitrogen loss and also serves to prevent contamination of the biological samples.
One way for achieving the required pressure differential limit is by varying the amount of refrigeration provided to the vessel interior by the cryocooler. As more liquid nitrogen is vaporized and the pressure within the vessel interior increases, the delivered refrigeration is increased to increase the nitrogen condensation rate and thus reduce the pressure. If the amount of nitrogen condensed increases to the point where the pressure within the vessel interior falls to below ambient pressure, the delivered refrigeration is decreased to allow the pressure within the vessel interior to approximate the ambient pressure.
Pressure relief conduit 26 has pressure relief valve 20 and communicates with the vessel interior. In the event the pressure within the vessel interior increases at a rate which cannot be addressed by the cryocooler operation described above, the pressure relief valve will open to reduce the pressure to the requisite level. The changes in the pressure relief valve opining, as well as changes in the cryocooler operation, can be controlled by reference to a pressure transducer such as differential pressure transducer 21 on conduit 26 in the embodiment of the invention illustrated in FIG. 1. The differential pressure transducer monitors the pressure difference between the vessel interior and the outside of the insulted vessel. In the embodiment illustrated in FIG. 1, equalizing valve 19 on pressure relief conduit 26 serves to equalize the pressure between the vessel interior and the outside when access to the vessel interior is desired by the removal of sealed lid 7.
Sealed lid 7 has pliable polymeric material in contact with the lid which serves to keep gas or vapor from flowing passed the lid, into or out of the vessel interior, when the pressure differential between the vessel interior and the outside of the vessel is within 2 psi. The polymeric material is maintained in compression while the lid 7 is positioned within opening 25. That is, the polymeric material is squeezed between two or more surfaces and is deformed so that it conforms to the space enclosing it. Among the polymeric materials which may be employed in the practice of this invention one can name silicone, polytetrafluoroethylene, ethylene chlorotrifluoroethylene, and ethylene propylene.
FIG. 2 illustrates one embodiment of a sealed lid which may be used in the practice of this invention. Referring now to FIG. 2, there is illustrated vacuum insulated neck 30 of the vessel with a screw thread ring 31 attached and sealed by polymeric thread ring seal 32. The lid foam insulation 33 is also shown which extends several inches below lid 7. The vessel thread ring may alternately be rolled into the inner wall of the vessel neck. The lid 7 is shown with a mating screw thread 34 and a polymeric lid seal gasket 35 on the inside wall. The threads may alternately be positioned on the outside wall. The lid seal gasket 35 prevents vapors from flowing past this seal within the pressure holding limits of the seal. The seal is kept in compression when the lid is closed by the force provided by the mating screw threads. This type of screw thread sealing system would preferably require only a quarter or half turn from the unengaged to the fully engaged positions. The thread is provided to produce the required compression for sealing of the lid gasket; however other compression techniques can be used. Of these other compression techniques, one can name clamping, tapered cork compression, magnetic compression or weighted compression systems.
Although the invention has been described in detail with reference to certain preferred embodiments, those skilled in the art will recognize that there are other embodiments of the invention within the spirit and the scope of the claims.

Claims

1. A cryogenic biological preservation unit comprising:

(A) an insulated vessel having a vessel interior, a lid positioned within an opening allowing access to the vessel interior, and a seal comprising pliable polymeric material in contact with the lid;

(B) a cryocooler positioned to provide refrigeration to the insulated vessel; and

(C) a pressure relief conduit having a pressure relief valve and being in communication with the vessel interior.

2. The cryogenic biological preservation unit of claim 1 wherein the cryocooler is a pulse tube refrigerator.

3. The cryogenic biological preservation unit of claim 1 containing at least one biological sample within the vessel interior.

4. The cryogenic biological preservation unit of claim 1 further comprising a differential pressure transducer on the pressure relief conduit.

5. A method for operating a cryogenic biological preservation unit comprising:

(A) providing refrigeration from a cryocooler to the interior of an insulated vessel, said insulated vessel having a lid and a seal comprising pliable polymeric material in contact with the lid;

(B) maintaining the pliable polymeric material in compression; and

(C) maintaining the pressure within the interior of the insulated vessel within 2 psi of the pressure outside of the insulated vessel.

6. The method of claim 5 wherein the pressure within the interior of the insulated vessel is maintained within 2 psi of the pressure outside of the insulated vessel by varying the amount of refrigeration provided from the cryocooler to the interior of the insulated vessel.

7. The method of claim 5 wherein the pliable polymeric material is maintained in compression by engaged mating screw threads on the lid and on an adjacent screw thread ring.