PREVENTING SAMPLE DEGRADATION FROM TRANSIENT TEMPERATURE FLUCTUATIONS

Abstract

Methods and devices can protect samples maintained at controlled temperatures from degradation due to transient temperature fluctuations.

Description

RELATED APPLICATIONS

This application claims priority to United States Provisional Patent Application Ser. No. 61/512,816, entitled PREVENTING SAMPLE DEGRADATION FROM TRANSIENT TEMPERATURE FLUCTUATIONS, filed on Jul. 28, 2011, which is incorporated herein by reference in its entirety.

BACKGROUND

The storage of sensitive laboratory samples and reagents in controlled temperature conditions, for example, in upright refrigerators or freezers or in incubators, presents numerous opportunities for temperature cycle-dependent degradation. Biological samples typically contain a complex solution that may include salts, buffering components, stabilizers, reducing agents, and cryo-protectants, in addition to organic isolates and purified macromolecular components. Repeated, transient temperature fluctuations of such samples during periods at which they are being held at controlled temperatures can damage them.

For example, during frozen storage, migration of smaller molecular species can occur at temperatures as low as −50° C. with the rate of migration increasing with temperature. The small molecule migrations can lead to focal changes in salt concentration, pH, and macromolecular hydration, leading to undesired effects, such as protein denaturation and aggregation. Although the samples may never become fully thawed during storage, cyclic variation in temperature will impose progressive damage to many types of biological samples, particularly to protein solutions such as cells and cellular components, such as preparations of antibodies, vaccines, and/or proteins with enzymatic activity. Proteins sensitive to degradation due to cyclic fluctuations in temperature of only 3° C. have been reported.

Under normal laboratory operation conditions, at least four sources of temperature fluctuation present a challenge to sample integrity of samples being held at controlled temperatures below freezing, and several of these sources are present in other temperature control devices, i.e., incubators that control temperatures at above room temperature and refrigerators that control temperatures at below room temperature and above freezing.

For freezers that control temperature in the range of 0° C. to −25° C., the first of these sources is the frost-free cycle of the freezer. During normal upright freezer access, the interior air, being at a lower temperature and greater density than the outside air, will flow out of the compartment to be replaced by warm and often moisture-laden air. Upon contact with the cold interior surfaces, the air is quickly reduced in temperature with a concurrent water vapor condensation on the cold surface, resulting in a net build-up of an ice layer. With time, the ice layer thickness can become substantial and can interfere with sample access and reduce the useful volume of the freezer interior. To prevent ice build-up, consumer-grade freezers and some laboratory grade freezers that are designed to operate in the −25° C. to 0° C. range commonly incorporate a regular frost-free cycle of approximately one hour in duration that warms the interior of the freezer and then returns the temperature to the normal temperature fluctuation cycle.

Although typically lower in amplitude than the frost-free cycle, a second and very regular source of cyclic temperature fluctuations in the freezer interior can result from the normal refrigeration compressor operation. The compressor cycle is controlled by a thermostatic regulator with an activation-deactivation temperature range set by the manufacturer. Exposed samples can follow the interior air temperature, closely resulting in a constant state of temperature fluctuation during storage. This problem exists not only with freezers that are designed to operate in the −25° C. to 0° C. range but with freezers that operate (control the temperature) at lower temperatures, i.e., −80° C. freezers.

The third source of temperature fluctuation derives from user access and can result in the most extreme temperature fluctuations. This problem exists with all temperature control devices, including incubators designed to control temperature at room temperature and above and refrigerators and freezers designed to control temperature at below room temperature. For example, an exchange of the cold interior air of a refrigerator or freezer for the warm external occurs rapidly upon opening of and upright freezer door. Unprotected samples, particularly those with low thermal mass to surface area ratios will rapidly adjust to the interior air temperature and will experience an increase in temperature as a result. This problem also exists with refrigerators. Laboratory freezers are often shared property with multiple users, which results in frequent opening of the freezer compartment to the outside air. The duration of an open door search for a specific item can easily result in an unintentional and severe rise in archived sample temperatures with warming to the point of visible melting of frost on container surfaces. In addition, co-workers that are unaware that the freezer has been recently accessed often compound the warming problem by near term subsequent accession of the freezer compartment.

The fourth source of undesired sample temperature fluctuation, i.e., warming in the case of a refrigerator or freezer or cooling in the case of an incubator, derives from the placement of room temperature samples in a storage location adjacent to samples already at the desired controlled temperature. For example, if a room temperature sample is placed next to previously frozen samples, then, as the thermal energy from the sample dissipates, the proximal frozen sample will transiently increase in temperature.

Current solutions to the temperature fluctuation problem include the inclusion of thermal mass panels in the freezer walls, and the storage of large thermal mass such as containers of frozen water in the freezer. While these solutions serve to return the freezer interior to the desired temperature more quickly upon closure, the low thermal conductivity of the air separating the samples from the thermal sinks delays the heat transfer from the samples, resulting in poor protection against transient spikes in temperature.

Other approaches to addressing the problem are tank racks with formed recesses to receive sample containers. The rack interiors contain aqueous gels to prevent leakage should the container rupture and to provide a phase change medium for the dual purpose of providing extended thermal protection of the samples outside the freezer. This solution has limitations in that the containers are subject to rupture and vulnerable to impact damage, and the relatively low density of the gels provides sub-optimal protection for a given rack volume. Therefore, rack dimensions must be increased or the sample storage capacity must be reduced to gain improvement in thermal protection.

Another approach to addressing the problem of sample temperature fluctuation is found in the form of insulated boxes; however, this approach has multiple negative features. An effective thickness of insulation can greatly reduce the usable storage volume. In addition, placement of room temperature samples in an insulated box will increase the length of time required to freeze the sample and subject all previously frozen samples to a spike in temperature as the thermal energy dissipates. As a result, samples have to be frozen outside the insulated box and placed into the box at a later time to avoid influencing the temperature of previously archived samples.

Thus, there remains a need in the art for methods and devices for preventing or otherwise retarding the degradation of frozen or chilled samples in freezers and refrigerators and for warmed samples in incubators. The present invention meets this need.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the present invention provides methods for preventing sample degradation of a sample in a container in a temperature control device due to transient temperature fluctuations, which method comprises placing the container into a thermoconductive container holder located in said refrigerator or freezer. In this method, the thermoconductive container is chilled (or warmed) to the desired controlled temperature prior to the sample container being placed in it. As those of skill in the art will appreciate, once the thermoconductive container holder is placed in the temperature control device, it is not removed therefrom (or otherwise not allowed to attain a temperature substantially different from the control temperature) in accordance with the methods of the invention.

In some embodiments of the method, the thermoconductive container holder is itself placed in another container, such as a cardboard box, a plastic box, or a container such as those marketed under the CoolBox™ and CoolCell® marks by BioCision (Mill Valley, Calif.).

The container holders useful in the methods of the invention are made of thermoconductive materials with heat transfer properties much better than plastic. Suitable thermoconductive materials have thermal conductivity values (k) measured at 68° Fahrenheit of at least 25, and optionally of at least 50 or even at least 100 Btu/(hr degrees F. ft) or greater. In various embodiments, the container holders are made from a metal or metal alloy, including but not limited to aluminum, i.e., anodized aluminum, copper, and aluminum alloys, particularly machinable aluminum alloys (i.e., those containing iron, magnesium, silicon, copper, zinc, manganese, chromium, titanium, and combinations thereof).

In a second aspect, the present invention provides effective solutions to the problems caused by transient temperature fluctuations in cooling devices such as refrigerators and freezers, by providing a means to suppress (minimize) those fluctuations in the chilled or frozen samples in those cooling devices. In various embodiments of the invention, the samples are contained in sample tubes, vials, or other commercially available laboratory sample holders, and the sample containers are placed in aluminum or aluminum alloy racks that are in the cooling device to practice the method of the invention. In a preferred embodiment, the sample is a frozen sample contained in a tube, the cooling device is a freezer designed to maintain temperatures less than 0° C., including but not limited to a −20° C. freezer, a −25° C. freezer, and a −80° C. freezer, and the container holder is made of aluminum or an aluminum alloy.

Those of skill in the art will appreciate, in view of this disclosure, that the higher density of the container holder, relative to that of the sample container, which is typically plastic, combined with the thermoconductive property of the material used to form the container holder results in the container holder acting as a very effective thermal stabilizer while minimizing the volume of the freezer space occupied by the container holder.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In order that the manner in which the above-recited and other features and advantages of the invention are obtained will be readily understood, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. These drawings depict only typical embodiments of the invention and are not therefore to be considered to limit the scope of the invention.

FIG. 1 is a perspective view of a container holder in accordance with a representative embodiment of the present invention.

FIG. 2 is a perspective view of a container holder and laboratory freezer enclosure or box in accordance with a representative embodiment of the present invention.

FIG. 3 is a cross-section view of a container holder in accordance with a representative embodiment of the present invention.

FIG. 4 is a chart demonstrating the temperature trace of exposed samples compared with samples enclosed within an insulated box in accordance with a representative embodiment of the present invention.

FIG. 5 is a chart demonstrating the temperature trace of samples in a thermoconductive rack compared with samples in an insulated box in accordance with a representative embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will be best understood by reference to the drawings, wherein like reference numbers indicate identical or functionally similar elements. It will be readily understood that the components of the present invention, as generally described and illustrated in the figures herein, could be arranged and designed in a wide variety of different configurations. Thus, the following more detailed description, as represented in the figures, is not intended to limit the scope of the invention as claimed, but is merely representative of presently preferred embodiments of the invention.

An embodiment of a container holder of the invention that is useful in the methods of the invention is shown in FIG. 1. In this embodiment, the container holder is a sample rack 110 constructed from a thermoconductive material having a thermal conductivity of approximately 170 watts per meter.degree Kelvin and a density of 2.7 grams per cubic centimeter, as can be fabricated from, for example, an aluminum alloy, and contains a plurality of wells 120 that will accept 64 sample vials 130 that have a maximum diameter of 0.5 inches and a height of approximately 2 inches. The rack dimensions measure 4.9 inches in both length and width with a height of 1.5 inches.

As shown in FIG. 2, the rack 230 is specifically designed to be enclosed in a standard cardboard laboratory freezer box 210 that has nominal dimensions of 5 inches by 5 inches by 2 inches, and is enclosed by a cardboard lid 220. The assembly 200 will fit within common standardized storage racks for laboratory freezers. As shown, the rack 230 is configured with 64 wells 240 that are designed to receive standard laboratory sample vials 250, but may also receive and store vials of lesser dimensions. The rack design shown in FIG. 1 is a representative example, and is not intended to limit the size of the rack, the number of sample wells, or the type or number of sample containers to be received.

A separate embodiment of the invention is shown in cross section in FIG. 3 (the CoolRack® 15 ml product marketed by BioCision, Mill Valley, Calif.). The rack 310 is constructed from the same or a similar material described for the rack in FIG. 1 and has dimensions of 3.49 inches in both length and width, a height of 4.4 inches, and a mass of 1.37 kg. The rack is designed to receive up to nine tubes 320 that have a maximum fill volume of 15 ml.

The effectiveness of the methods of the invention in suppressing temperature fluctuation is demonstrated in FIGS. 4 and 5.

In FIG. 4, a thermocouple was used to monitor the interior air temperature of a freezer compartment set by the manufacturer to an average temperature of −20° C. (dashed line). Numerous refrigeration compressor cycles can be observed with a period of approximately 0.8 hours and with a temperature range of −25° C. to −17° C. In addition, two frost-free cycles occur during the 32 hour interval at approximately 4 hours and 21 hours with a temperature range of −25° C. to −8° C. The temperature of a 5 ml volume of water contained within a 15 ml maximum volume screw-cap sample tube when placed exposed on a laboratory shelf closely followed the interior air temperature throughout the measurement interval (solid grey line). The temperature of an identical sample contained in a polyethylene foam insulated box (the CoolBox™ device marketed by BioCision) with a wall thickness of 1 inch is recorded in the solid black line. In this trace, it is seen that the insulation is effective in partially suppressing the degree of fluctuation in the sample temperature.

In FIG. 5, again the temperature trace for an exposed thermocouple probe showing the interior air temperature is shown in the dashed line. Fluctuations in temperature again cycled between −25° C. and −17.5° C. during the compressor cycle and −25° C. to −8.5° C. during the frost-free cycle, which occurred at 15 hours. At approximately 3 hours, the freezer door was opened for 30 seconds and again at approximately 19 and 26 hours, after which the interior temperature reached 1° C. for the first accession and −8.5° C. and −7.5° C. for the later two accession, respectively. The temperature of a 5 ml sample of water contained in a 15 ml sample tube and placed within a foam box identical to that used in FIG. 4 was again effective in partially suppressing the temperature fluctuation range of the sample (solid grey trace (labeled “CoolBox tube temp” in the legend)); however, when an additional room temperature sample was placed into the insulated box at 19 hours and 26 hours, a more severe temperature increase was observed than was seen when no additional sample was introduced at 4 hours. In addition, the temperature increase persisted through the following compressor cycle, demonstrating that the heat contained in the introduced sample influenced the temperature of the monitored sample.

The temperature of an identical sample tube placed in the rack described in FIG. 3 is shown by the black solid trace in FIG. 5 (labeled “CoolRack tube temp” in the legend). Although the average temperature of the sample in the rack was one degree warmer than the average temperature of the sample in the foam box (−19.5° C. versus −20.5° C.), the range of temperature fluctuation was significantly reduced by comparison. Introduction of a second room temperature sample into a well adjacent to the monitored sample at 19 and 26 hours resulted is a substantially smaller increase in the monitored sample compared to the increase observed with the sample in the foam box.

Those of skill in the art will appreciate, in view of this disclosure, that the methods and devices of the invention are effective in suppressing temperature fluctuations in samples and that the suppressive property is independent of the average temperature of the environment. Therefore, these methods and devices are effective in suppressing temperature fluctuations in a warm incubator, for example in the range of 0° to 100° C., as well as in refrigerators (15° C. to 0° C., i.e., 4° C.) and freezers (below 0° C.).

The methods of the invention can be practiced with any device composed of materials with the properties described herein. In various embodiments, the device is a device marketed under the CoolRack® mark (BioCision, Mill Valley, Calif.). See also, U.S. patent application publication No. 20090258407, incorporated herein by reference, for other container holders useful in the methods of the invention.

Those of skill in the art will appreciate, in view of this disclosure, that the methods described herein are especially useful in maintaining frozen samples in freezers with freeze thaw cycling and/or in freezers that are frequently accessed. In addition, the methods of the invention enable samples to be stored safely (without substantial degradation) in such freezers for time periods far longer than that achievable by prior art methods and devices, i.e., for at least 3 months, at least 6 months, at least a year, at least 5 years, and at least 10 years or longer.

Practicing the methods of the invention can result in significantly longer stability of samples and improvements in sample stability in relatively short periods of time. For example, enzymes, such as restriction enzymes, are typically stored in commercially available freezers that utilize freeze-thaw cycles for frost control (other frost-free freezers are much more expensive). Application of the methods and use of the devices of the invention enables these enzymes to be maintained with much less loss of activity in such freezers, with remarkable improvements achievable, depending on frequency of access and other factors, within 1, 3, and 6 months of initial storage. For more expensive materials, such as cell lines and cell banks, even where freezers that do not utilize freeze-thaw cycles (i.e., more expensive freezers or freezers that operate at temperatures below −25° C.) and even where access is less frequent, the methods and devices of the invention can provide remarkable improvement in stability over the relevant time periods, i.e., within 1, 5, and 10 years or longer.

The present invention may be embodied in other specific forms without departing from its structures, methods, or other essential characteristics as broadly described herein and claimed hereinafter. The described embodiments are to be considered in all respects only as illustrative, and not restrictive. The scope of the invention is, therefore, indicated by the appended claims, rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

1. A method for preventing or reducing sample degradation of a sample in a sample container in a controlled temperature device subject to transient temperature fluctuations over a period of at least one month, which method comprises placing the sample container into a thermoconductive container holder having a thermal conductivity of at least about 150 watts per meter.degree Kelvin and a density of at least about 2.5 grams per cubic centimeter in such device.

2. The method of claim 1, wherein such device is a refrigerator or freezer.

3. The method of claim 1, wherein the sample is a biological sample.

4. The method of claim 3, wherein the device is a freezer, and the controlled temperature is below freezing.

5. The method of claim 4, wherein the sample is a living cell.

6. The method of claim 4, wherein the sample is a protein or protein extract.

7. The method claim 1, wherein the container holder is composed of a material with a thermal conductivity greater than 10 Watts per meter.degree Kelvin.

8. The method of claim 7, wherein the material is an aluminum alloy.

9. The method of claim 8, wherein the material has a density of greater than 1 gram per cubic centimeter.

10. The method of claim 9, wherein the container holder will fit into a space having a width and length of at least 5 ×5 inches and a height of at least 1.5 inches.