Patent Application
20150072424
1. Field of the Invention
The present invention relates in general to both a cryogenic cooling thin film evaporator and a method for using same.
2. Description of Related Art
Cryogenic cooling is used in a variety of applications, including but not limited to cell and embryo cryopreservation, cryosurgery, cryoelectronics and cryotronics, cryobiology, cryofixation and cryosubstitution for electron microscopy, and cryogenics.
One prevalent method of cell cryopreservation is vitrification, a method which is regarded as a preferred alternative to the traditional slow freezing method. Vitrification, a process in which both cells and extracellular solutions are solidified without ice formation, is typically achieved by a “pool boiling” procedure. In a “pool boiling” procedure, samples are directly plunged or dropped into a pool of coolant that is typically liquid nitrogen. Cells generally are not in direct contact with liquid nitrogen; rather, they are either surrounded by solutions or some layers of polymers, thus forming a “sample.” Vitrification offers two advantages over the traditional slow freezing method. First, vitrification causes the cell temperature to drop more rapidly through the temperature range where intracellular and extracellular ice crystals can form than the traditional slow freezing method, resulting in reduced cell mechanical injury (or avoiding cell injury altogether). Second, vitrification methods eliminate the need for the time consuming and laborious cooling procedures involved in slow cooling methods.
In a conventional vitrification procedure using pooling boiling cooling methods, the typical cooling rate of cells is approximately 2,500° C./min. The use of an electron microscope (EM) grip in a pool boiling method can increase the cooling rate to 24,000° C./min. The use of open pulled straws (OPS) in a pool boiling method can increase the cooling rate to 20,000° C./min. Likewise, the use of a cryoloop in a pool boiling method can increase the cooling rate to 20,000° C./min. Although these methods achieve complete or partial vitrification of cell suspensions, highly concentrated cryoprotectants are required, resulting in reduced cell survival rates due to the toxicity of the cryoprotectant.
The primary reason to date that the cooling rate for vitrification methods has not been increased and there is a dependence on the use of higher concentrations of cryoprotectants is that liquid nitrogen vaporizes near the surface of the samples while the samples are submerged in the liquid nitrogen. The evaporating nitrogen forms a heat insulating layer referred to as a “vapor blanket.” As a result, the heat transfer coefficient between the sample surface and the liquid nitrogen is limited (less than 103 W/m2K) due to the poor thermal conduction of the “vapor blanket.” Therefore, removal of the “vapor blanket” is critical in order to further improve upon the cooling rate of conventional vitrification.
The present invention is directed in a first aspect to an apparatus for the cryogenic cooling of a substance. The apparatus includes a microstructured surface and an applicator for dispensing a working fluid onto the microstructured surface. In one aspect of the invention, the microstructured surface includes micropores. The micropores may have a diameter between 1 and 100 microns in diameter. The micropores may be formed from microparticles, which may have a diameter between 1 and 500 microns. In another aspect of the invention, the microstructured surface may be formed from microstructures such as nanowires, etched microchannels and microfabricated microstructures. In one aspect of the invention, the microstructured surface may be made of a metal selected from the group consisting of copper, gold, silver, iron, aluminum, nickel, platinum, and their oxides. In one aspect of the invention, the total thickness of the microstructured surface may range from 1 to 1000 microns.
In one aspect of the invention, the microstructured surface may include a base that supports the microstructured surface. In one such aspect, the base may have a thickness ranging from 10 to 1000 microns. In one such aspect, the base may be formed from a metal selected from the group consisting of copper, gold, silver, iron, aluminum, nickel, platinum, and their oxides. In one aspect, the base is formed from the same metal as the microstructured surface.
In one aspect of the invention, the apparatus may also include a pressure-controlled vessel that encloses the thin film evaporator and at least a portion of said applicator. In one such aspect, the pressure of the pressure-controlled vessel is controlled by a vacuum operably connected to the vessel.
In another aspect of the invention, the present invention is directed to a method for using an apparatus of the present invention. In one such embodiment, the apparatus comprises a thin film evaporator having a first microporous surface and a second surface opposite the microporous surface. In one such aspect, a substance to be cooled is placed adjacent to the second surface. In one such aspect, a working fluid is then dispensed onto the microstructured surface such that the working fluid forms a thin liquid film. In one aspect, the working fluid may be liquid nitrogen, liquid helium, liquid oxygen, or liquid oxygen. In one aspect, the thickness of the thin liquid film formed is between 1 and 100 microns. In one aspect of the present invention, the pressure of the thin film evaporator is lowered from a first pressure to a second pressure that is below the saturation pressure of the working fluid prior to dispensing the working fluid onto the microstructured surface. In one such aspect, the pressure lowering step may be performed by a vacuum.
In one embodiment, the method of the second aspect of the present invention is performed such that the thin film evaporator is enclosed in a pressure-controlled vessel during the pressure lowering step wherein the pressure of the thin film evaporator is lowered from a first pressure to a second pressure. In one such aspect, the pressure of the pressure-controlled vessel may be maintained at the second pressure of the thin film evaporator below the saturation pressure of the working fluid while the working fluid is being dispensed onto the microstructured surface.
In one aspect of the invention, the method of the present invention may be used to cool substances including but not limited to living cells, living embryos, or living thin tissues.
Additional aspects of the invention, together with the advantages and novel features appurtenant thereto, will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned from the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
a-1) depicts a cross-sectional view of one exemplary embodiment of the thin film evaporator of the present invention.
a-2) depicts a top-plan view of one exemplary embodiment of the thin film evaporator of the present invention.
b) depicts a 3D photograph of an exemplary microstructured surface of one embodiment of the present invention, produced in Example 1.
c) is a horizontal cross-sectional view of an exemplary microstructured surface of one embodiment of the present invention, produced in Example 1.
One aspect of the present invention is directed to an apparatus for cryogenically cooling a substance. The apparatus of the present invention includes a thin film evaporator comprising a microstructured surface and an applicator for dispensing a working fluid onto the microstructured surface. The apparatus preferably contains a pressure-controlled vessel enclosing the thin film evaporator and at least a portion of the applicator. In a second aspect, the present invention is directed to the method of using a thin film evaporator of the present invention to cryogenically cool a substance by forming a thin film layer of the working fluid on the microstructured surface of the thin film evaporator.
Looking to
Microstructured surface 12 may be comprised of any structures that form pores consistent with the present invention. Structures forming the microstructured surface 12 may include microparticles, nanowires, etched microchannels, and microfabricated microstructures.
In the embodiment wherein microstructured surface 12 is comprised of microparticles, as depicted in
Micro structured surface 12 may be comprised of any material suitable for forming microstructures consist with the present invention. In one embodiment, the microstructured surface is formed from a metal, preferably a metal that may be sintered, formed into nanowires, etched or microfabricated microstructures. The metal may be selected from the group consisting of copper, gold, silver, iron, aluminum, nickel, and platinum. Copper, gold, and silver are particularly well-suited for use in the present invention. In one embodiment, the microstructured surface is formed from a metal oxide, preferably a metal oxide that may be sintered, formed into nanowires, etched or microfabricated microstructures. The metal oxide may be selected from the group consisting of copper oxide, gold oxide, silver oxide, iron oxide, aluminum oxide, nickel oxide, and platinum oxide.
In one embodiment of the invention, microstructured surface 12 is between 1 and 1000 microns thick, and in one such embodiment is between 10 and 200 microns thick. In one exemplary embodiment, the microstructured surface is 150 microns thick. The thickness of microstructured surface 12 will vary depending on the specific application and use.
Base 14 of thin film evaporator 10, if present, may be formed of any material described above with respect to microstructured surface 12. Base 14 and microstructured surface 12 may be comprised of the same material or different materials. Base 14 may be any thickness capable of supporting microstructured surface 12. In one embodiment, base 14 is thinner than microstructured surface 12. In one such embodiment base 14 is between 10 and 1000 microns thick, or between 100 and 200 microns thick. In one exemplary embodiment, base 14 is 50 microns thick. In one embodiment, the total thickness of thin film evaporator 10, including both microstructured surface 12 and base 14 is between 10 and 1000 microns thick, or between 100 and 500 microns thick.
Although the thin film evaporator is generally planar in the exemplary embodiment depicted in
The shape and dimension of the thin film evaporator may vary depending on the substance to be cryogenically cooled. In certain embodiments the thin film evaporator will be a rectangular, square or rounded shape but is not limited to such shapes.
In the exemplary embodiment shown in
Pressure-controlled vessel 28 may have a pressure sensor or pressure gauge 32 operably connected to it that is used to monitor and measure the pressure within pressure-controlled vessel 28. A thermocouple 34 may also be attached to the back of thin film evaporator 10 (i.e., the side opposite microstructured surface 12 which is referred to as second surface 42 below) to monitor and measure its temperature over time. A data acquisition (“DAQ”) system 36 connected to a computer 38 may also be connected to pressure sensor 32 and thermocouple 34 to record the temperature and pressure. DAQ 36 and computer 38 may also be operably connected to valve 26 and/or a vacuum valve 40 to regulate the vacuum and liquid flow applied to pressure-controlled vessel 28. In a commercial setting, DAQ 36, computer 38, pressure sensor 32, and thermocouple 34 may be entirely omitted.
The present invention is also directed to a method for using the thin film evaporator of the present invention. In one such embodiment, a thin film evaporator according to the present invention is provided. Thin firm evaporator 10 comprises a first microstructured surface 12 and a second surface 42 opposite the first micro structured surface. Second surface 42 may be a surface of microstructured surface 10 opposite a first surface of microstructured surface 10. Alternatively, if base 14 is included, second surface 42 is the surface of base 14 opposite microstructured surface 12. A substance to be cryogenically cooled is placed adjacent to second surface 42. As used herein, “adjacent” includes direct application of the substance to second surface 42, as well as placement of the substance sufficiently near second surface 42 to come into contact with the thin liquid film (as described below). Preferably the substance is placed within 1 to 100 microns of second surface 42. In one embodiment, the substance is applied adjacent to second surface 42 such that it is of uniform thickness so that the cooling of the substance occurs as uniformly and evenly as possible.
A working fluid is then dispensed to contact microstructured surface 12 such that the working fluid forms a thin liquid film 44 along at least a portion of microstructured surface 12. The working fluid will preferably spread along the entire surface of microstructured surface 12, and into micropores 16, due to the capillary force produced by the microstructured surface. Microstructured surface 12 produces capillary force and disjoining pressure to form the thin liquid film on the microstructured surface. This in turn prevents the microstructured surface from drying out. Importantly, it also reduces the formation of a vapor blanket which allows an ultra-fast cooling rate to be achieved. In one embodiment the thin liquid film is between 1 and 100 microns thick, and in one such embodiment between 1 and 10 microns thick.
The presence of the numerous microscale pores in microstructured surface 12 increases the surface area over that which would be offered by a smooth, flat surface, thereby increasing the total evaporating thin film region. Additionally, the thermal resistance across the thin liquid film is very small because the evaporating thin film is very thin, resulting in an increased heat transfer coefficient. The increased heat transfer coefficient causes the evaporating surface temperature to fall rapidly from room temperature to the saturation temperature. In one embodiment, the heat transfer coefficient between the sample surface and the thin film is at least 107W/m2K, and in one such embodiment at least 105W/m2K.
The time required to fall from a first starting temperature to a second cooling temperature can be less than 0.1 seconds and in one such embodiment is between 0.2 and 0.3 seconds. The second cooling temperature can be readily determined based on the intended use of the apparatus and process of the invention. In one embodiment, wherein the apparatus is used for cryopreservation, the second temperature is lower than −190° C., and in one such embodiment between −196° C. and −190° C. The average cooling rate can exceed 105 ° C./minute and in one embodiment is between 104 and 105° C./minute. In such an embodiment, the average cooling rate was observed to be more than three times higher than the cooling rate observed using standard vitrification techniques.
The working fluid may be any liquid that vaporizes at cryogenic temperatures. The working fluid may be but is not limited to liquid helium, liquid nitrogen, liquid oxygen, liquid argon, and many others. The working fluid is applied at a mass flow rate sufficient to keep maintain a thin film of liquid working fluid on the microstructured surface. The vacuum pump or other pressure regulator will affect the required mass flow rate, as will be readily understood by one of ordinary skill in the art. In one exemplary embodiment, the mass flow rate of liquid nitrogen as the working gas is 1.934 g/s.
In one embodiment, the pressure surrounding the thin film evaporator is lowered from a first pressure to a second pressure below the saturation pressure of the working fluid, preferably prior to the dispensing step. When the thin film evaporator is at room temperature, the relevant saturation pressure is the saturation pressure at room temperature. Ideally, the pressure of the pressure-controlled vessel is maintained such that the vessel is as close to a pure vacuum as possible. In one exemplary embodiment where nitrogen is the working fluid, the pressure may be lowered to 500 Pa. A vacuum pump is preferably used to lower the pressure, although other pressure regulators may be used. In the embodiment wherein the thin film evaporator is enclosed within a pressure-controlled vessel, the pressure of the vessel is lowered so that it is lower than the saturation pressure of the working fluid. The second pressure within the vessel is preferably maintained below the saturation pressure of the working fluid during the dispensing step. In the exemplary embodiment shown in
The working fluid is then dispensed onto microstructured surface 12 via an applicator as discussed above. The further below the saturation pressure at room temperature of the working fluid that the pressure of the vessel is maintained, the higher the rate of evaporation of the working fluid will be and the faster that molecules of the working fluid that are in the vapor phase will move away from the liquid-vapor interface of the working fluid and toward the vapor within the vessel. This in turn causes the temperature at the liquid-vapor interface to decrease at a faster rate.
The method of the present invention may be used to cryogenically cool substances including but not limited to living cells, living embryos, and living thin tissues. The method of the present invention may be used in various applications in which cryogenic cooling is desirable. In addition the cryopreservation of biological tissue discussed herein, other applications include cryosurgery, cryofixing in electron microscopy, cryogenic thermal management that can be used in spacecraft or other environments, and cryogenic machining.
The following examples are directed to various exemplary embodiments of the apparatus and method of the present invention and their use in accordance with the present invention.
A pressure-controlled vessel was connected to a vacuum pump to control the pressure and to enhance the evaporation of liquid nitrogen. The generally planar thin film evaporator with a microstructured surface is depicted in
Prior to the start of the experiment, the pressure of the pressure-controlled vessel was brought to an absolute pressure of approximately 500 Pa. The valve on the liquid nitrogen container was then opened. Liquid nitrogen flowed from the liquid nitrogen container and through the needle to the generally planar thin film evaporator. As liquid nitrogen reached the point of application of the microstructured surface, the liquid spread along the remainder of the microstructured surface due to the capillary force produced by the microscale pores of the microstructures shown in
The vacuum pump was continually operated during the experiment and removed some of the nitrogen vapor within the pressure-controlled vessel. The pressure within the pressure-controlled vessel rose from approximately 500 Pa to approximately 190,670 Pa as shown in
In order to compare the cryogenic cooling performance of the generally planar thin film evaporator as used in Experiment 1 (thin film evaporation) with a pool boiling process, a comparative experiment in which the generally planar thin film evaporator was plunged directly into liquid nitrogen was also performed.
From the foregoing it will be seen that this invention is one well adapted to attain all ends and objectives herein-above set forth, together with the other advantages which are obvious and which are inherent to the invention.
Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matters herein set forth or shown in the accompanying drawings are to be interpreted as illustrative, and not in a limiting sense.
While specific embodiments have been shown and discussed, various modifications may of course be made, and the invention is not limited to the specific forms or arrangement of parts and steps described herein, except insofar as such limitations are included in the following claims. Further, it will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.
This application is based on and claims priority to U.S. Provisional Application Ser. No. 61/960,111 filed on Sep. 10, 2013, which is incorporated herein by reference in its entirety.
This invention was made with Government support under Grant No.1 R21 RR025908-02 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61960111 | Sep 2013 | US |
